RESUMO
The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient's course of the underlying disease.
Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Antígeno de Maturação de Linfócitos B/metabolismo , Sistemas Automatizados de Assistência Junto ao Leito , Imunoterapia Adotiva/métodos , Mieloma Múltiplo/patologia , Citocinas/metabolismo , Linfócitos T , CriopreservaçãoRESUMO
The transcription factor SOX11 is a tumor-associated antigen with low expression in normal cells, but overexpression in glioblastoma (GBM). So far, conventional surgery, chemotherapy, and radiotherapy have not substantially improved the dismal prognosis of relapsed/refractory GBM patients. Immunotherapy is considered a promising strategy against GBM, but there is a fervent need for better immunotargets in GBM. To this end, we performed an in silico prediction study on SOX11, which primarily yielded ten promising HLA-A*0201-restricted peptides derived from SOX11. We defined a novel peptide FMACSPVAL, which had the highest score according to in silico prediction (6.02 nM by NetMHC-4.0) and showed an exquisite binding affinity to the HLA-A*0201 molecule in the peptide-binding assays. In the IFN-γ ELISPOT assays, FMACSPVAL demonstrated a high efficiency for generating SOX11-specific CD8+ T cells. Nine out of thirty-two healthy donors showed a positive response to SOX11, as assessed by the ELISPOT assays. Therefore, this novel antigen peptide epitope seems to be promising as a target for T cell-based immunotherapy in GBM. The adoptive transfer of in vitro elicited SOX11-specific CD8+ T cells constitutes a potential approach for the treatment of GBM patients.
Assuntos
Glioblastoma , Glioma , Humanos , Linfócitos T CD8-Positivos , Epitopos de Linfócito T , Glioma/metabolismo , Glioblastoma/metabolismo , Peptídeos/química , Imunoterapia , Linfócitos T Citotóxicos , Fatores de Transcrição SOXC/metabolismoRESUMO
Extracorporeal photopheresis (ECP), a personalized cellular immunotherapy, constitutes a promising treatment for steroid-refractory/-resistant graft-versus-host disease (SR-GvHD), with encouraging clinical response rates. To further investigate its mechanism of action, ECP's effects on T helper (Th) cells as well as on expression of immune checkpoint (PD-1 and Tim-3) and apoptotic (Fas receptor [FasR]) molecules were investigated in 27 patients with SR-GvHD. Our data show that GvHD patients had significantly higher levels of Th2, Th17, Th22 and granulocyte-macrophage colony-stimulating factor (GM-CSF)-positive Th (ThG) cells and clearly lower levels of T follicular helper (Tfh) cells, including Th1- and Th2-like cells, compared with healthy donors. ECP therapy for GvHD was effective through the modulation of different Th subsets: increases of Th22 (1.52-fold) and Tfh cells (1.48-fold) in acute GvHD (aGvHD) and increases of Th2-like Tfh cells (1.74-fold) in chronic GvHD (cGvHD) patients were associated with clinical response. Expression of FasR was further upregulated in CD4+CD8+ T cells. Additionally, Tim-3-expressing effector T cells associated with the severity of GvHD were reduced. Taken together, these data show that ECP therapy exerts immunomodulatory effects by promoting a balanced immune reconstitution and inducing immune tolerance. Therefore it represents an attractive option for the treatment of GvHD.
Assuntos
Doença Enxerto-Hospedeiro , Fotoferese , Linfócitos T CD8-Positivos , Doença Crônica , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Esteroides/uso terapêutico , Células T Auxiliares Foliculares , Regulação para CimaRESUMO
After solid-organ transplantation, reactivation of the cytomegalovirus (CMV) is often observed in seronegative patients and associated with a high risk of disease and mortality. CMV-specific T cells can prevent CMV reactivation. In a phase 1 trial, CMV-seronegative patients with end-stage renal disease listed for kidney transplantation were subjected to CMV phosphoprotein 65 (CMVpp65) peptide vaccination and further investigated for T-cell responses. To this end, CMV-specific CD8+ T cells were characterized by bulk T-cell-receptor (TCR) repertoire sequencing and combined single-cell RNA and TCR sequencing. In patients mounting an immune response to the vaccine, a common SYE(N)E TCR motif known to bind CMVpp65 was detected. CMV-peptide-vaccination-responder patients had TCR features distinct from those of non-responders. In a non-responder patient, a monoclonal inflammatory T-cell response was detected upon CMV reactivation. The identification of vaccine-induced CMV-reactive TCRs motifs might facilitate the development of cellular therapies for patients wait-listed for kidney transplantation.
Assuntos
Infecções por Citomegalovirus/prevenção & controle , Falência Renal Crônica/terapia , Receptores de Antígenos de Linfócitos T/genética , Proteínas da Matriz Viral/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Ensaios Clínicos Fase I como Assunto , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/administração & dosagem , Vacinas contra Citomegalovirus/imunologia , Humanos , Falência Renal Crônica/imunologia , Transplante de Rim , Análise de Sequência de RNA , Imagem Individual de Molécula , Proteínas da Matriz Viral/imunologiaRESUMO
Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.
Assuntos
Antígeno de Maturação de Linfócitos B/metabolismo , Citometria de Fluxo , Reação em Cadeia da Polimerase , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Antígeno de Maturação de Linfócitos B/genética , Biomarcadores , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos Quiméricos/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Linfócitos T/imunologiaRESUMO
Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B-cell lymphoma or acute lymphoblastic leukemia. Impaired T-cell fitness of CLL patients may be involved in treatment failure. Less-differentiated naïve-like T cells play an important role in CART expansion and long-term persistence in vivo. These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B-cell receptor inhibitor ibrutinib targeting Bruton's tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin-2-inducible T-cell kinase (ITK) which is involved in T-cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells from nine healthy donors and eight CLL patients were used to generate CART cells. T-cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient-derived CART cells. Furthermore, ibrutinib enriched CART cells with less-differentiated naïve-like phenotype and decreased expression of exhaustion markers including PD-1, TIM-3 and LAG-3. In addition, ibrutinib increased the cytokine release capacity of CLL patient-derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient-derived CART cell products.
Assuntos
Adenina/análogos & derivados , Imunoterapia Adotiva/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Piperidinas/farmacologia , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Adenina/farmacologia , Antígenos CD19/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Meios de Cultura , Citocinas/biossíntese , Células HEK293 , Humanos , Células K562 , Leucemia Linfocítica Crônica de Células B/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted.
Assuntos
Imunoterapia Adotiva/métodos , Leucemia Linfocítica Crônica de Células B/imunologia , Purinas/farmacologia , Quinazolinonas/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Interleucina-15/farmacologia , Interleucina-17/farmacologia , Leucemia Linfocítica Crônica de Células B/sangue , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos de Linfócitos T/biossíntese , Linfócitos T/imunologiaRESUMO
The anti-tumor efficacy of TCR-engineered T cells in vivo depends largely on less-differentiated subsets such as T cells with naïve-like T cell (TN) phenotypes with greater expansion and long-term persistence. To increase these subsets, we compared the generation of New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cells under supplementation with either IL-2 or IL-7/IL-15. PBMCs were transduced with MS3II-NY-ESO-1-siTCR retroviral vector. T cell generation was adapted from a CD19-specific CART cell production protocol. Comparable results in viability, expansion and transduction efficiency of T cells under stimulation with either IL-2 or IL-7/IL-15 were observed. IL-7/IL-15 led to an increase of CD4+ T cells and a decrease of CD8+ T cells, enriched the amount of TN among CD4+ T cells but not among CD8+ T cells. In a 51Cr release assay, similar specific lysis of NY-ESO-1-positive SW982 sarcoma cells was achieved. However, intracellular cytokine staining revealed a significantly increased production of IFN-γ and TNF-α in T cells generated by IL-2 stimulation. To validate these unexpected findings, NY-ESO-1-specific T cell production was evaluated in another protocol originally established for TCR-engineered T cells. IL-7/IL-15 increased the proportion of TN. However, the absolute number of TN did not increase due to a significantly slower expansion of T cells with IL-7/IL-15. In conclusion, IL-7/IL-15 does not seem to be superior to IL-2 for the generation of NY-ESO-1-specific T cells. This is in sharp contrast to the observations in CD19-specific CART cells. Changes of cytokine cocktails should be carefully evaluated for individual vector systems.
Assuntos
Antígenos de Neoplasias/metabolismo , Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Proteínas de Membrana/metabolismo , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Antígenos CD19/metabolismo , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Meios de Cultura , Humanos , Interleucina-15/imunologia , Interleucina-2/imunologia , Interleucina-7/imunologia , Proteínas de Membrana/imunologia , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/genéticaRESUMO
BACKGROUND: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. METHODS: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. RESULTS: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DISCUSSION: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.
Assuntos
Criopreservação/métodos , Receptores de Antígenos Quiméricos/genética , Linfócitos T/transplante , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Transplante de Células/métodos , Radioisótopos de Cromo/análise , Radioisótopos de Cromo/metabolismo , Citocinas/metabolismo , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Controle de Qualidade , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologiaRESUMO
Chimeric antigen receptor T cell (CART) therapy is currently one of the most promising treatment approaches in cancer immunotherapy. However, the immunosuppressive nature of the tumor microenvironment, in particular increased reactive oxygen species (ROS) levels, provides considerable limitations. In this study, we aimed to exploit increased ROS levels in the tumor microenvironment with prodrugs of ROS accelerators, which are specifically activated in cancer cells. Upon activation, ROS accelerators induce further generation of ROS. This leads to an accumulation of ROS in tumor cells. We hypothesized that the latter cells will be more susceptible to CARTs. CD19-specific CARTs were generated with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Cytotoxicity was determined by chromium-51 release assay. Influence of the ROS accelerators on viability and phenotype of CARTs was determined by flow cytometry. The combination of CARTs with the ROS accelerator PipFcB significantly increased their cytotoxicity in the Burkitt lymphoma cell lines Raji and Daudi, as well as primary chronic lymphocytic leukemia cells. Exposure of CARTs to PipFcB for 48 h did not influence T cell exhaustion, viability, or T cell subpopulations. In summary, the combination of CARTs with ROS accelerators may improve adoptive immunotherapy and help to overcome tumor microenvironment-mediated treatment resistance.
Assuntos
Leucemia de Células B/imunologia , Leucemia de Células B/metabolismo , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Degranulação Celular , Linhagem Celular Tumoral , Citocinas/biossíntese , Citotoxicidade Imunológica , Humanos , Leucemia de Células B/patologia , Leucemia de Células B/terapia , Linfoma de Células B/patologia , Linfoma de Células B/terapia , Estresse Oxidativo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genéticaRESUMO
Tumor-mediated immunosuppression via regulatory T-cells is a key player among the various immune-escape mechanisms in multiple myeloma. We analyzed the generation, distribution, function and immunophenotype of CD8+CD28- regulatory T-cells in patients with multiple myeloma. Functionality of CD8+CD28- T-cells was assessed by immunological assays using ex vivo generated antigen-specific T-cells from patients with plasma cell dyscrasias and healthy donors. Detailed analysis of distribution, immunophenotype and cytotoxic potential of CD8+CD28- T-cells was performed by flow cytometry and ELISA. We found that the amount of CD8+CD28- T-cells was directly correlated with the suppression of antigen-specific T-cell responses in patients with plasma cell dyscrasia. Analyzing the CD8+CD28- T-cells in detail, increased numbers of these cells were observed in the bone marrow (i.e., tumor microenvironment) of patients with plasma cell dyscrasia. Furthermore, we identified the expression of lymphocyte function-associated antigen 1 (LFA-1) as a marker of immunosuppression and defined the CD8+CD28-CD57+LFA-1high population as the relevant immunosuppressive compartment. These regulatory T-cells act as immunosuppressors via soluble factors and incubation with IL-10 augmented their immunosuppressive capacity. The immunosuppressive regulatory network of IL-10 and the CD8+CD28-CD57+LFA-1high regulatory T-cells show unique characteristics and contribute to the tumor immune escape mechanism in patients with multiple myeloma.
Assuntos
Antígenos CD28/imunologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-10/farmacologia , Mieloma Múltiplo/imunologia , Paraproteinemias/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Humanos , Ativação Linfocitária , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Paraproteinemias/metabolismo , Paraproteinemias/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
BACKGROUND: Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties. METHODS: iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings. RESULTS: In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses. CONCLUSION: In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT.
Assuntos
Doença Enxerto-Hospedeiro , Células T Matadoras Naturais , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Receptor de Morte Celular Programada 1 , Antígenos CD19 , Doença Enxerto-Hospedeiro/etiologia , RecidivaRESUMO
ABSTRACT: Graft-versus-host disease (GVHD) occurs in about 10% to 33% of patients receiving "allogeneic" or "autologous" chimeric antigen receptor T (CAR-T) cells after preceding allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to the substantial presence of alloreactive T cells. Extracorporeal photopheresis (ECP) shows promising clinical outcomes in the treatment of GVHD after allo-HSCT without hampering antitumor and antiviral effects. This raises an interesting question: whether ECP might constitute a new way to treat patients with GVHD after CAR T-cell therapy without compromising CAR-T cells significantly. Third-generation CD19-specific CAR-T cells were generated and an in vitro ECP protocol was established. The impact of ECP on CAR-T cells was comprehensively investigated in 2 models: the nondilution model reflects days after CAR T-cell infusion and the dilution model weeks after infusion. The therapeutic effect of ECP on GVHD was examined in an in vitro mixed lymphocyte reaction (MLR) assay. We found, ECP-treated CAR-T cells demonstrated reduced potency in inducing alloreaction compared with that of the group without ECP treatment in MLR assay. ECP could selectively induce apoptosis, thereby enriching the naive and central memory CAR-T cells with a reduced alloreactivity. The cytokine milieu of CAR-T cells could be switched from immune stimulation to immune tolerance in both models. Moreover, ECP could modulate the proliferative capacity of CAR-T cells without hampering their long-term functionality in the dilution model. In conclusion, ECP constitutes a promising treatment strategy for GVHD after allo-HSCT and CAR T-cell transfusion, as ECP reduces the alloreactivity without hampering CAR T-cell functionality.
Assuntos
Doença Enxerto-Hospedeiro , Imunoterapia Adotiva , Fotoferese , Doença Enxerto-Hospedeiro/terapia , Doença Enxerto-Hospedeiro/etiologia , Fotoferese/métodos , Humanos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/efeitos adversos , Receptores de Antígenos Quiméricos , Linfócitos T/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodosRESUMO
The aim of this study was to investigate whether the specific T cell response against the multiple myeloma Ag HM1.24 is enhanced by the immunomodulatory drug lenalidomide (Revlimid). Ag-specific CD3(+)CD8(+) T cells against the HM1.24 Ag were expanded in vitro by dendritic cells in 29 healthy donors and 26 patients with plasma cell dyscrasias. Ag-specific activation was analyzed by IFN-γ, granzyme B, and perforin secretion using ELISA, ELISPOT assay, and intracellular staining, and generation of Ag-specific T cells was analyzed by tetramer staining. Expression of T cell maturation markers (CD45RA, CD45R0, CCR7, and CD28) was investigated by flow cytometry. We found that activation of HM1.24-specific T cells from healthy donors and patients with plasma cell dyscrasias was enhanced significantly by lenalidomide and furthermore that the impact of lenalidomide on T cells depends on the duration of the exposure. Notably, lenalidomide supports the downregulation of CD45RA on T cells upon activation, observed in healthy donors and in patients in vitro and also in patients during lenalidomide therapy in vivo. We showed for the first time, to our knowledge, that lenalidomide enhances the Ag-specific activation of T cells and the subsequent downregulation of CD45RA expression of T cells in vitro and in vivo.
Assuntos
Regulação para Baixo/imunologia , Epitopos de Linfócito T/metabolismo , Antígenos Comuns de Leucócito/antagonistas & inibidores , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Subpopulações de Linfócitos T/imunologia , Talidomida/análogos & derivados , Regulação para Cima/imunologia , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Epitopos de Linfócito T/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lenalidomida , Antígenos Comuns de Leucócito/biossíntese , Ativação Linfocitária/imunologia , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Paraproteinemias/imunologia , Paraproteinemias/metabolismo , Paraproteinemias/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Talidomida/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) Tcells. In order to overcome a lottolot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serumfree media [PrimeXV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ TCell Expansion XenoFree Medium, Takara Bio™ (TB) and TCM GMPPrototype, CellGenix™ (CG)] to the standard CAR Tcell medium containing FBS (RCF). After 12 days of CD19.CAR Tcell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR Tcells was evaluated using intracellular staining, a chromium release assay and a longterm coculture assay. Expansion and viability did not differ between the CAR Tcells generated in serumfree media compared to the standard FBScontaining medium. The CG CAR Tcells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNFα+ CAR Tcells than the CAR Tcells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR Tcells generated with serumfree media exhibited a higher cytotoxicity than the CAR Tcells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR Tcell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyteactivation gene 3 and Tcell immunoglobulin and mucindomain containing3. The CAR Tcells cultured in FF had a higher percentage of central memory CAR Tcells (40.0 vs. 14.3%, P=0.0470) than the CAR Tcells cultured with FBS, whereas the CAR Tcells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR Tcells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serumfree media. Given the advantages of freedom from bovine material and consistent quality, serumfree media hold promise for the future development of the field of GMP manufacturing of CAR Tcells.
Assuntos
Citocinas , Linfócitos T , Animais , Humanos , Meios de Cultura Livres de Soro/metabolismo , Linfócitos T/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , CromoRESUMO
BACKGROUND: Third-generation chimeric antigen receptor (CAR)-engineered T cells (CARTs) might improve clinical outcome of patients with B cell malignancies. This is the first report on a third-generation CART dose-escalating, phase-1/2 investigator-initiated trial treating adult patients with refractory and/or relapsed (r/r) acute lymphoblastic leukemia (ALL). METHODS: Thirteen patients were treated with escalating doses of CD19-directed CARTs between 1 × 106 and 50 × 106 CARTs/m2. Leukapheresis, manufacturing and administration of CARTs were performed in-house. RESULTS: For all patients, CART manufacturing was feasible. None of the patients developed any grade of Immune effector cell-associated neurotoxicity syndrome (ICANS) or a higher-grade (≥ grade III) catokine release syndrome (CRS). CART expansion and long-term CART persistence were evident in the peripheral blood (PB) of evaluable patients. At end of study on day 90 after CARTs, ten patients were evaluable for response: Eight patients (80%) achieved a complete remission (CR), including five patients (50%) with minimal residual disease (MRD)-negative CR. Response and outcome were associated with the administered CART dose. At 1-year follow-up, median overall survival was not reached and progression-free survival (PFS) was 38%. Median PFS was reached on day 120. Lack of CD39-expression on memory-like T cells was more frequent in CART products of responders when compared to CART products of non-responders. After CART administration, higher CD8 + and γδ-T cell frequencies, a physiological pattern of immune cells and lower monocyte counts in the PB were associated with response. CONCLUSION: In conclusion, third-generation CARTs were associated with promising clinical efficacy and remarkably low procedure-specific toxicity, thereby opening new therapeutic perspectives for patients with r/r ALL. Trial registration This trial was registered at www. CLINICALTRIALS: gov as NCT03676504.
Assuntos
Síndromes Neurotóxicas , Humanos , Adulto , Leucaférese , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD19/uso terapêuticoRESUMO
Adoptive cell therapy with NY-ESO-1-specific T cells is a promising option for the treatment of soft tissue sarcoma (STS) but achieves only transient tumor control in the majority of cases. A strategy to optimize this cell therapeutic approach might be the modulation of the expression of the cancer-testis antigen NY-ESO-1 using histone deacetylase inhibitors (HDACis). In this study, the ex vivo effect of combining NY-ESO-1-specific T cells with the clinically approved pan HDACis panobinostat or vorionstat was investigated. Our data demonstrated that STS cells were sensitive to HDACis. Administration of HDACi prior to NY-ESO-1-specific T cells exerted enhanced lysis against the NY-ESO-1+ STS cell line SW982. This correlated with an increase in the NY-ESO-1 and HLA-ABC expression of SW982 cells, as well as increased CD25 expression on NY-ESO-1-specific T cells. Furthermore, the immune reactivity of NY-ESO-1-specific CD8+ T cells in terms of cytokine release was enhanced by HDACis. In summary, pretreatment with HDACis represents a potential means of enhancing the cytotoxic efficacy of NY-ESO-1-specific T cells against NY-ESO-1-positive STS.
RESUMO
Chimeric antigen receptor (CAR) T cell therapy with axicabtagene ciloleucel, tisagenlecleucel and brexucabtagen ciloleucel has been adopted as the standard of care for patients with refractory and/or relapsed CD19positive lymphoid malignancies. Monitoring of kinetics of CAR T cells after administration is crucial for patient followup and important to guide clinical decisions for patients subjected to CAR T cell therapy. Information of transgene copies within a CAR T cell product prior to administration, i.e. vector copy numbers, is of high importance to warrant patient safety. However, experimental assays for quantitative CAR T cell monitoring in the open domain are currently lacking. Several institutions have established inhouse assays to monitor CAR T cell frequencies. In the present study, the quantitative (q)PCR assay established at the Heidelberg University Hospital (Heidelberg, Germany), i.e. single copy genebased duplex qPCR, was compared with the digital droplet PCR assay established at the University Medical Center HamburgEppendorf (Hamburg, Germany). Both methods that were independently developed enable accurate and comparable CAR T cell frequency assessment and are useful in the clinical setting.
Assuntos
Antígenos CD19/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Imunoterapia Adotiva , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/imunologia , Biomarcadores/sangue , Progressão da Doença , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
INTRODUCTION: Donor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in a cellular phenotype that was directly and indirectly suppressive to the recipient's immune system allowing for reduction of conventional immunosuppressive therapy. Here, we describe a protocol for a randomised controlled, multicentre phase-IIb clinical trial of individualised immunosuppression with intravenously administered donor MIC compared with standard-of-care (SoC) in living donor kidney transplantation (TOL-2 Study). METHODS AND ANALYSIS: Sixty-three living donor kidney transplant recipients from six German transplant centres are randomised 2:1 to treatment with MIC (MIC group, N=42) or no treatment with MIC (control arm, N=21). MIC are manufactured from donor peripheral blood mononuclear cells under Good Manufacturing Practice conditions. The primary objective of this trial is to determine the efficacy of MIC treatment together with reduced conventional immunosuppressive therapy in terms of achieving an operational tolerance-like phenotype compared with SoC 12 months after MIC administration. Key secondary endpoints are the number of patient-relevant infections as well as a composite of biopsy-proven acute rejection, graft loss, graft dysfunction or death. Immunosuppressive therapy of MIC-treated patients is reduced during follow-up under an extended immunological monitoring including human leucocyte antigen-antibody testing, and determination of lymphocyte subsets, for example, regulatory B lymphocytes (Breg) and antidonor T cell response. A Data Safety Monitoring Board has been established to allow an independent assessment of safety and efficacy. ETHICS AND DISSEMINATION: Ethical approval has been provided by the Ethics Committee of the Medical Faculty of the University of Heidelberg, Heidelberg, Germany (AFmu-580/2021, 17 March 2022) and from the Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institute, Langen, Germany (Vorlage-Nr. 4586/02, 21 March 2022). Written informed consent will be obtained from all patients and respective donors prior to enrolment in the study. The results from the TOL-2 Study will be published in peer-reviewed medical journals and will be presented at symposia and scientific meetings. TRIAL REGISTRATION NUMBER: NCT05365672.
Assuntos
Transplante de Rim , Humanos , Transplante de Rim/efeitos adversos , Doadores Vivos , Padrão de Cuidado , Leucócitos Mononucleares , Terapia de Imunossupressão , Imunossupressores/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Multicêntricos como Assunto , Ensaios Clínicos Fase II como AssuntoRESUMO
Chimeric antigen receptor (CAR) T cells directed against CD19 (CD19.CAR T cells) have yielded impressive clinical responses in the treatment of patients with lymphoid malignancies. However, resistance and/or relapse can limit treatment outcome. Risk of tumor escape can be reduced by combining treatment strategies. Selective inhibitors of nuclear export (SINEs) directed against nuclear exportin1 (XPO1) have demonstrated antitumor efficacy in several hematological malignancies. The aim of the present study was to evaluate the combination of CAR T cells with the SINE compounds eltanexor and selinexor. As expected, eltanexor and selinexor were toxic to CD19positive malignant cells and the sensitivity of cells towards SINEs correlated with the levels of XPO1expression in ALL cell lines. When SINEs and CAR T cells were simultaneously combined, SINEs exerted toxicity towards CAR T cells and impaired their function affecting cytotoxicity and cytokine release ability. Flow cytometry and western blot analysis revealed that eltanexor decreased the cytoplasmic concentration of the transcription factor phosphorylatedSTAT3 in CAR T cells. Due to CAR Tcell toxicity, sequential use of SINEs and CAR T cells was evaluated: Cytotoxicity of CAR T cells increased significantly when target cells were pretreated with the SINE compound eltanexor. In addition, exhaustion of CAR T cells decreased when target cells were pretreated with eltanexor. In summary, whereas the concomitant use of SINEs and CAR T cells does not seem advisable, sequential use of SINEs and CAR T cells might improve the antitumor efficacy of CAR T cells.