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1.
Methods Mol Biol ; 2853: 17-31, 2025.
Artículo en Inglés | MEDLINE | ID: mdl-39460912

RESUMEN

Fusion proteins are valuable molecules to meet different demands related to the development of biopharmaceuticals and bioprocesses. In human therapy, they are used to improve the half-life of biologics by modifying the biophysical properties of the proteins. In biotechnology, the design of fusion proteins can standardize the establishment of production clones and the purification process. Analytical detection capabilities of the fusion partner and binding to affinity ligands play a crucial role. For the generation of the recombinant cell line, the maturation of the protein and the secretion are also crucial factors, which can be significantly influenced by the fusion partner and can determine the final yield of the bioprocess. Here we present a protocol to recombine the human extracellular domain of CD19 with the human serum albumin domain 2 resulting in a fusion protein CD19-AD2 including a flexible linker sequence in the interface between the C-terminus of CD19 and the N-terminus of HSA-D2 and a terminal His12-tag. The two fragments are independently amplified with primers allowing to genetically connect the two fragments in the next step by overlap extension PCR. By this strategy, the linker sequence as well as the albumin fragment can be chosen individually to be flexible in the fine-tuning of the final protein. The amplified product is then cloned into a mammalian expression vector suitable to generate a recombinant transient or stable cell culture. This workflow can be applied to any protein sequence by adapting the specific primer sequences.


Asunto(s)
Antígenos CD19 , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Antígenos CD19/genética , Antígenos CD19/metabolismo , Clonación Molecular/métodos , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo
2.
Front Immunol ; 15: 1415328, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39192973

RESUMEN

Background: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. Methods: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Results: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. Conclusions: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.


Asunto(s)
Antígenos CD19 , Elementos Transponibles de ADN , Inmunoterapia Adoptiva , Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/métodos , Antígenos CD19/inmunología , Antígenos CD19/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/genética , Células Nutrientes , Linfoma de Células B/terapia , Linfoma de Células B/inmunología , Linfoma de Células B/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Electroporación , Células Alogénicas/inmunología
3.
Mol Ther ; 32(8): 2676-2691, 2024 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-38959896

RESUMEN

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.


Asunto(s)
Anticuerpos Biespecíficos , Antígenos CD19 , Células Plasmáticas , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Anticuerpos Biespecíficos/farmacología , Animales , Ratones , Antígenos CD19/inmunología , Antígenos CD19/genética , Antígenos CD19/metabolismo , Células Plasmáticas/metabolismo , Células Plasmáticas/inmunología , Línea Celular Tumoral , Linfocitos T/inmunología , Linfocitos T/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Complejo CD3/genética , Activación de Linfocitos/inmunología , Citotoxicidad Inmunológica
4.
Front Immunol ; 15: 1401683, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38868778

RESUMEN

Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.


Asunto(s)
Antígenos CD19 , Sistemas CRISPR-Cas , Edición Génica , Memoria Inmunológica , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Transcriptoma , Humanos , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/efectos adversos , Antígenos CD19/inmunología , Antígenos CD19/genética , Edición Génica/métodos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Fenotipo , Línea Celular Tumoral
6.
Hepatobiliary Pancreat Dis Int ; 23(5): 472-480, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-38724321

RESUMEN

BACKGROUND: Regulatory B cells (Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs (miRNAs), miR-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and miR-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs (mBregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. METHODS: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce miR-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. RESULTS: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of mBregs in the circulating blood were significantly impaired. miR-29a-3p was found to be a regulator of mBregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5 (NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of mBregs. The inhibition of miR-29a-3p in CD19+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into mBregs. In addition, the observed enhancement of differentiation and immunosuppressive function of mBregs upon miR-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. CONCLUSIONS: miR-29a-3p was found to be a crucial regulator for mBregs differentiation and immunosuppressive function. Silencing miR-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.


Asunto(s)
Antígenos CD19 , Linfocitos B Reguladores , Antígeno CD24 , Diferenciación Celular , Trasplante de Hígado , MicroARNs , Humanos , MicroARNs/metabolismo , MicroARNs/genética , Linfocitos B Reguladores/inmunología , Linfocitos B Reguladores/metabolismo , Antígenos CD19/metabolismo , Antígenos CD19/genética , Masculino , Antígeno CD24/metabolismo , Antígeno CD24/genética , Transducción de Señal , Rechazo de Injerto/inmunología , Rechazo de Injerto/genética , Femenino , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Persona de Mediana Edad , Tolerancia Inmunológica , Células Cultivadas , Adulto , Fenotipo , Memoria Inmunológica
7.
J Immunother Cancer ; 12(4)2024 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-38677881

RESUMEN

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.


Asunto(s)
Antígenos CD19 , Antígenos CD20 , Elementos Transponibles de ADN , Inmunoterapia Adoptiva , Regiones Promotoras Genéticas , Receptores Quiméricos de Antígenos , Antígenos CD19/inmunología , Antígenos CD19/genética , Humanos , Animales , Antígenos CD20/genética , Antígenos CD20/metabolismo , Antígenos CD20/inmunología , Ratones , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Inmunoterapia Adoptiva/métodos , Ratones Endogámicos NOD , Línea Celular Tumoral , Ratones SCID , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Sci Signal ; 17(826): eadd4671, 2024 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-38442200

RESUMEN

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)-mediated signaling, but the number and type of protein interaction-mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Transducción de Señal , Antígenos CD19/genética , Membrana Celular , Receptores de Antígenos de Linfocitos T/genética
9.
J Transl Med ; 22(1): 274, 2024 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-38475814

RESUMEN

BACKGROUND: Chimeric antigen receptor natural killer (CAR-NK) cells represent a promising advancement in CAR cell therapy, addressing limitations observed in CAR-T cell therapy. However, our prior study revealed challenges in CAR-NK cells targeting CD19 antigens, as they failed to eliminate CD19+ Raji cells in NSG tumor-bearing mice, noting down-regulation or loss of CD19 antigen expression in some Raji cells. In response, this study aims to enhance CD19 CAR-NK cell efficacy and mitigate the risk of tumor recurrence due to target antigen escape by developing CD19 and CD20 (CD19/CD20) dual-targeted CAR-NK cells. METHODS: Initially, mRNA encoding anti-CD19 CARs (FMC63 scFv-CD8α-4-1BB-CD3ζ) and anti-CD20 CARs (LEU16 scFv-CD8α-4-1BB-CD3ζ) was constructed via in vitro transcription. Subsequently, CD19/CD20 dual-targeted CAR-NK cells were generated through simultaneous electrotransfection of CD19/CD20 CAR mRNA into umbilical cord blood-derived NK cells (UCB-NK). RESULTS: Following co-electroporation, the percentage of dual-CAR expression on NK cells was 86.4% ± 1.83%, as determined by flow cytometry. CAR expression was detectable at 8 h post-electric transfer, peaked at 24 h, and remained detectable at 96 h. CD19/CD20 dual-targeted CAR-NK cells exhibited increased specific cytotoxicity against acute lymphoblastic leukemia (ALL) cell lines (BALL-1: CD19+CD20+, REH: CD19+CD20-, Jurkat: CD19-CD20-) compared to UCB-NK, CD19 CAR-NK, and CD20 CAR-NK cells. Moreover, CD19/CD20 dual-targeted CAR-NK cells released elevated levels of perforin, IFN-γ, and IL-15. Multiple activation markers such as CD69 and cytotoxic substances were highly expressed. CONCLUSIONS: The creation of CD19/CD20 dual-targeted CAR-NK cells addressed the risk of tumor escape due to antigen heterogeneity in ALL, offering efficient and safe 'off-the-shelf' cell products. These cells demonstrate efficacy in targeting CD20 and/or CD19 antigens in ALL, laying an experimental foundation for their application in ALL treatment.


Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores Quiméricos de Antígenos , Ratones , Animales , Receptores Quiméricos de Antígenos/metabolismo , Antígenos CD19/genética , Antígenos CD19/metabolismo , Citotoxicidad Inmunológica/genética , Línea Celular Tumoral , Células Asesinas Naturales , Inmunoterapia Adoptiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , ARN Mensajero/metabolismo
10.
J Clin Invest ; 134(8)2024 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-38376944

RESUMEN

While therapies targeting CD19 by antibodies, chimeric antigen receptor T cells (CAR-T), and T cell engagers have improved the response rates in B cell malignancies, the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19 immunotoxin. Single-cell RNA-Seq (scRNA-Seq) showed an increase in transcriptionally distinct CD19lo populations among resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19lo-resistant cells. An assay for transposase-accessible chromatin with sequencing (ATAC-Seq) showed decreased chromatin accessibility at promoters of both CD19 and CD22 in the resistant cell populations. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with B cell acute lymphoblastic leukemia (B-ALL) that relapsed after CD19 CAR-T-targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19lo resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both Bruton's tyrosine kinase (BTK) and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.


Asunto(s)
Antígenos CD19 , Leucemia-Linfoma Linfoblástico de Células Precursoras , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Niño , Humanos , Adulto Joven , Agammaglobulinemia Tirosina Quinasa , Antígenos CD19/genética , Cromatina , Inmunoterapia Adoptiva , Quinasas de Proteína Quinasa Activadas por Mitógenos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores Quiméricos de Antígenos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética
11.
Cell Rep Med ; 5(2): 101421, 2024 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-38340727

RESUMEN

Chimeric antigen receptor T cell (CAR T) therapy is a potent treatment for relapsed/refractory (r/r) B cell lymphomas but provides lasting remissions in only ∼40% of patients and is associated with serious adverse events. We identify an upregulation of CD80 and/or CD86 in tumor tissue of (r/r) diffuse large B cell lymphoma (DLBCL) patients treated with tisagenlecleucel. This finding leads to the development of the CAR/CCR (chimeric checkpoint receptor) design, which consists of a CD19-specific first-generation CAR co-expressed with a recombinant CTLA-4-linked receptor with a 4-1BB co-stimulatory domain. CAR/CCR T cells demonstrate superior efficacy in xenograft mouse models compared with CAR T cells, superior long-term activity, and superior selectivity in in vitro assays with non-malignant CD19+ cells. In addition, immunocompetent mice show an intact CD80-CD19+ B cell population after CAR/CCR T cell treatment. The results reveal the CAR/CCR design as a promising strategy for further translational study.


Asunto(s)
Linfoma de Células B Grandes Difuso , Linfocitos T , Humanos , Animales , Ratones , Antígeno CTLA-4 , Linfoma de Células B Grandes Difuso/terapia , Linfoma de Células B Grandes Difuso/etiología , Inmunoterapia Adoptiva/métodos , Linfocitos B , Antígenos CD19/genética
12.
J Immunol Methods ; 525: 113603, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-38147898

RESUMEN

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19. Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19+ Nalm-6-GFP cells and CD19- Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19+ but not CD19- 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells. Therefore, CD19+ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.


Asunto(s)
Receptores Quiméricos de Antígenos , Ratones , Animales , Humanos , Receptores Quiméricos de Antígenos/genética , Inmunoterapia Adoptiva , Pruebas Inmunológicas , Activación de Linfocitos , Antígenos CD19/genética
13.
Autoimmunity ; 56(1): 2281225, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-38053370

RESUMEN

OBJECTIVE: High-throughput sequencing was used to screen expressing differences of miRNA, lncRNA, and mRNA in CD19+ B peripheral blood samples of newly diagnosed immune thrombocytopenia (ITP) patients and healthy controls. The study aimed to explore the regulatory role of ceRNA network in the pathogenesis of dysfunctional CD19 + B lymphocytes of ITP patients. METHODS: CD19+ B lymphocytes were isolated from peripheral blood samples of ITP patients and their healthy counterparts. High-throughput sequencing was used to screen for the expression of miRNA, lncRNA, and mRNA of ITP patients and healthy controls, which were analysed by the ceRNA network. Moreover, qPCR was used to verify the differential expression of miRNA, lncRNA, and mRNA in ITP patients and healthy controls. The correlation between differentially expressed miRNA, lncRNA, mRNA, and B lymphocyte subsets was also analysed. RESULTS: The CD19+ B lymphocytes of 4 newly diagnosed ITP patients and 4 healthy controls were sequenced and analysed. There were 65 differentially expressed lncRNA and 149 mRNA forming a ceRNA network showed that 12 lncRNA and 136 differentially expressed mRNA were closely associated. Similarly, miR-144-3p, miR-374c-3p, and miR-451a were highly expressed in ITP patients, as confirmed by qPCR, which was consistent with the high-throughput sequence results. LOC102724852 and CCL20 were highly expressed in ITP patients, while LOC105378901, LOC112268311, ALAS2, and TBC1D3F were not as compared to healthy controls, which was consistent with the high-throughput sequence results. In addition, the expression of miR-374c-3p, LOC112268311, LOC105378901, and CXCL3 were correlated with the percentage of B lymphocyte subsets. CONCLUSIONS: The ceRNA network of miRNA, lncRNA, and mRNA in peripheral CD19 + B lymphocytes plays an essential role in the pathogenesis of ITP.


Asunto(s)
MicroARNs , Púrpura Trombocitopénica Idiopática , ARN Largo no Codificante , Trombocitopenia , Humanos , Púrpura Trombocitopénica Idiopática/diagnóstico , Púrpura Trombocitopénica Idiopática/genética , ARN Largo no Codificante/genética , MicroARNs/genética , Linfocitos B , ARN Mensajero/genética , Antígenos CD19/genética , Redes Reguladoras de Genes , 5-Aminolevulinato Sintetasa/genética
14.
Curr Oncol ; 30(12): 10463-10476, 2023 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-38132396

RESUMEN

Chimeric antigen receptor T (CAR T)-cell therapy has become a standard treatment option for patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Mutations in the PPM1D gene, a frequent driver alteration in clonal hematopoiesis (CH), lead to a gain of function of PPM1D/Wip1 phosphatase, impairing p53-dependent G1 checkpoint and promoting cell proliferation. The presence of PPM1D mutations has been correlated with reduced response to standard chemotherapy in lymphoma patients. In this study, we analyzed the impact of low-frequency PPM1D mutations on the safety and efficacy of CD19-targeted CAR T-cell therapy in a cohort of 85 r/r DLBCL patients. In this cohort, the prevalence of PPM1D gene mutations was 20% with a mean variant allele frequency (VAF) of 0.052 and a median VAF of 0.036. CAR T-induced cytokine release syndrome (CRS) and immune effector cell-associated neuro-toxicities (ICANS) occurred at similar frequencies in patients with and without PPM1D mutations. Clinical outcomes were globally worse in the PPM1D mutated (PPM1Dmut) vs. PPM1D wild type (PPM1Dwt) subset. While the prevalent treatment outcome within the PPM1Dwt subgroup was complete remission (56%), the majority of patients within the PPM1Dmut subgroup had only partial remission (60%). Median progression-free survival (PFS) was 3 vs. 12 months (p = 0.07) and median overall survival (OS) was 5 vs. 37 months (p = 0.004) for the PPM1Dmut and PPM1Dwt cohort, respectively. Our data suggest that the occurrence of PPM1D mutations in the context of CH may predict worse outcomes after CD19-targeted CAR T-cell therapy in patients with r/r DLBCL.


Asunto(s)
Linfoma de Células B Grandes Difuso , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva/efectos adversos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/uso terapéutico , Linfoma de Células B Grandes Difuso/terapia , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Resultado del Tratamiento , Antígenos CD19/genética , Antígenos CD19/uso terapéutico , Proteína Fosfatasa 2C/genética
15.
Int Immunopharmacol ; 124(Pt B): 110974, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37757633

RESUMEN

CAR-T targeting CD19 have achieved significant effects in the treatment of B-line leukemia and lymphoma. However, the treated patients frequently relapsed and could not achieve complete remission. Therefore, improving the proliferation and cytotoxicity of CAR-T cells, reducing exhaustion and enhancing infiltration capacity are still issues to be solved. The IL-7 has been shown to enhance the memory characteristics of CAR-T cells, but the specific mechanism has yet to be elaborated. miRNAs play an important role in T cell activity. However, whether miRNA is involved in the activation of CAR-T cells by IL-7 has not yet been reported. Our previous study had established the 3rd generation CAR-T cells. The present study further found that IL-7 significantly increased the proliferation of anti-CD19 CAR-T cells, the ratio of CD4 + CAR + cells and the S phase of cell cycle. In vivo study NAMALWA xenograft model showed that IL-7-stimulated CAR-T cells possessed stronger tumoricidal efficiency. Further we validated that IL-7 induced CAR-T cells had low expression of CDKN1A and high expression of miRNA-98-5p. Additionally, CDKN1A was associated with miRNA-98-5p. Our results, for the first time, suggested IL-7 could conspicuously enhance the proliferation of CAR-T cells through miRNA-98-5p targeting CDKN1A expression, which should be applied to CAR-T production.


Asunto(s)
MicroARNs , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Interleucina-7/genética , Interleucina-7/metabolismo , MicroARNs/genética , Proliferación Celular , Antígenos CD19/genética , Antígenos CD19/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo
16.
CPT Pharmacometrics Syst Pharmacol ; 12(9): 1285-1304, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37448297

RESUMEN

Chimeric antigen receptor (CAR) T-cell subsets and immunophenotypic composition of the pre-infusion product, as well as their longitudinal changes following infusion, are expected to affect CAR T-cell expansion, persistence, and clinical outcomes. Herein, we sequentially evolved our previously described cellular kinetic-pharmacodynamic (CK-PD) model to incorporate CAR T-cell product-associated attributes by utilizing published preclinical and clinical datasets from two affinity variants (FMC63 and CAT19 scFv) anti-CD19 CAR T-cells. In step 1, a unified cell-level PD model was used to simultaneously characterize the in vitro killing datasets of two CAR T-cells against CD19+ cell lines at varying effector:target ratios. In step 2, an augmented CK-PD model for anti-CD19 CAR T-cells was developed, by integrating CK dataset(s) from two bioanalytical measurements (quantitative polymerase chain reaction and flow cytometry) in patients with cancer. The model described the differential in vivo expansion properties of CAR T-cell subsets. The estimated expansion rate constant was ~1.12-fold higher for CAR+CD8+ cells in comparison to CAR+CD4+ T-cells. In step 3, the model was extended to characterize the disposition of four immunophenotypic populations of CAR T-cells, including stem-cell memory, central memory, effector memory, and effector cells. The model adequately characterized the longitudinal changes in immunophenotypes post anti-CD19 CAR T-cell infusion in pediatric patients with acute lymphocytic leukemia. Polyclonality in the pre-infusion product was identified as a categorical covariate influencing differentiation of immunophenotypes. In the future, this model could be leveraged a priori toward optimizing the composition of CAR T-cell infusion product, and further understand the CK-PD relationship in patients.


Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores Quiméricos de Antígenos , Humanos , Niño , Receptores Quiméricos de Antígenos/metabolismo , Cinética , Subgrupos de Linfocitos T/metabolismo , Inmunoterapia Adoptiva , Antígenos CD19/genética , Antígenos CD19/metabolismo , Receptores de Antígenos de Linfocitos T
17.
Nat Med ; 29(7): 1700-1709, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37407840

RESUMEN

In the context of relapsed and refractory childhood pre-B cell acute lymphoblastic leukemia (R/R B-ALL), CD19-targeting chimeric antigen receptor (CAR)-T cells often induce durable remissions, which requires the persistence of CAR-T cells. In this study, we systematically analyzed CD19 CAR-T cells of 10 children with R/R B-ALL enrolled in the CARPALL trial via high-throughput single-cell gene expression and T cell receptor sequencing of infusion products and serial blood and bone marrow samples up to 5 years after infusion. We show that long-lived CAR-T cells developed a CD4/CD8 double-negative phenotype with an exhausted-like memory state and distinct transcriptional signature. This persistence signature was dominant among circulating CAR-T cells in all children with a long-lived treatment response for which sequencing data were sufficient (4/4, 100%). The signature was also present across T cell subsets and clonotypes, indicating that persisting CAR-T cells converge transcriptionally. This persistence signature was also detected in two adult patients with chronic lymphocytic leukemia with decade-long remissions who received a different CD19 CAR-T cell product. Examination of single T cell transcriptomes from a wide range of healthy and diseased tissues across children and adults indicated that the persistence signature may be specific to long-lived CAR-T cells. These findings raise the possibility that a universal transcriptional signature of clinically effective, persistent CD19 CAR-T cells exists.


Asunto(s)
Leucemia Linfocítica Crónica de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Antígenos CD19/genética , Inmunoterapia Adoptiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfocitos T , Inducción de Remisión , Linfocitos T
18.
J Clin Immunol ; 43(7): 1543-1556, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37246174

RESUMEN

BACKGROUND: The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear. OBJECTIVE: Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model. METHODS: Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells. RESULTS: Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation. CONCLUSION: CD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.


Asunto(s)
Antígenos CD19 , Células Plasmáticas , Humanos , Células Plasmáticas/metabolismo , Antígenos CD19/genética , Antígenos CD19/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Linfocitos B , Receptores de Antígenos de Linfocitos B , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo
19.
Ann Hematol ; 102(6): 1523-1535, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37086278

RESUMEN

Chimeric antigen receptor T cell therapy (CAR-T) is a novel treatment that has produced unprecedented clinical effects in patients with hematological malignancies. Acute adverse events often occur following adoptive immunotherapy. Therefore, a suicide gene is helpful, which is a genetically encoded mechanism that allows selective destruction of adoptively transferred T cells in the face of unacceptable toxicity. RQR8 is a gene that integrates CD34 and CD20 epitopes. In our study, we incorporated the suicide gene RQR8 into CAR-T cells, so it enabled rituximab to eliminate vector/transgene-expressing T cells via antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity. In this work, we explored the functionality of RQR8 CAR-T cells in vitro and in vivo. We believe that RQR8 as a safety switch will make CAR-T cell therapy safer and less costly.


Asunto(s)
Receptores Quiméricos de Antígenos , Linfocitos T , Humanos , Receptores Quiméricos de Antígenos/genética , Inmunoterapia Adoptiva , Rituximab , Apoptosis , Antígenos CD19/genética
20.
Nat Biotechnol ; 41(11): 1606-1617, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36849828

RESUMEN

Chimeric antigen receptor T cell (CAR-T) expansion and persistence vary widely among patients and predict both efficacy and toxicity. However, the mechanisms underlying clinical outcomes and patient variability are poorly defined. In this study, we developed a mathematical description of T cell responses wherein transitions among memory, effector and exhausted T cell states are coordinately regulated by tumor antigen engagement. The model is trained using clinical data from CAR-T products in different hematological malignancies and identifies cell-intrinsic differences in the turnover rate of memory cells and cytotoxic potency of effectors as the primary determinants of clinical response. Using a machine learning workflow, we demonstrate that product-intrinsic differences can accurately predict patient outcomes based on pre-infusion transcriptomes, and additional pharmacological variance arises from cellular interactions with patient tumors. We found that transcriptional signatures outperform T cell immunophenotyping as predictive of clinical response for two CD19-targeted CAR-T products in three indications, enabling a new phase of predictive CAR-T product development.


Asunto(s)
Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores de Antígenos de Linfocitos T/genética , Inmunoterapia Adoptiva , Linfocitos T , Antígenos CD19/genética
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