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1.
Bull Cancer ; 2021 Jul 09.
Artigo em Francês | MEDLINE | ID: mdl-34253335

RESUMO

CAR-T cells represent a new anti-tumor immunotherapy which has shown its clinical efficacy in B-cell malignancies. The results of clinical trials carried out in this context have shown that certain immunological characteristics of patients before (at the time of apheresis) and after the administration of the treatment, or of the CAR-T cells themselves, are correlated with the response to the treatment or to its toxicity. However, to date, there are no recommendations on the immunological monitoring of patients treated in real life. The objectives of this workshop were to determine, based on data from the literature and the experience of the centers, the immunological analyses to be carried out in patients treated with CAR-T cells. The recommendations relate to the characterization of the patient's immune cells at the time of apheresis, the characterization of the injected CAR-T cells, as well as the monitoring of the CAR-T cells and other parameters of immune reconstitution in the patient after administration of the treatment. Harmonization of practices will allow clinical-biological correlation studies to be carried out in patients treated in real life with the aim of identifying factors predictive of response and toxicity. Such data could have a major medico-economic impact by making it possible to identify the patients who will optimally benefit from these expensive treatments.

2.
J Transl Med ; 19(1): 265, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34154602

RESUMO

BACKGROUND: Genetically engineered chimeric antigen receptor (CAR) T lymphocytes are promising therapeutic tools for cancer. Four CAR T cell drugs, including tisagenlecleucel (tisa-cel) and axicabtagene-ciloleucel (axi-cel), all targeting CD19, are currently approved for treating B cell malignancies. Flow cytometry (FC) remains the standard for monitoring CAR T cells using a recombinant biotinylated target protein. Nevertheless, there is a need for additional tools, and the challenge is to develop an easy, relevant, highly sensitive, reproducible, and inexpensive detection method. Molecular tools can meet this need to specifically monitor long-term persistent CAR T cells. METHODS: Based on 2 experimental CAR T cell constructs, IL-1RAP and CS1, we designed 2 quantitative digital droplet (ddPCR) PCR assays. By targeting the 4.1BB/CD3z (28BBz) or 28/CD3z (28z) junction area, we demonstrated that PCR assays can be applied to approved CD19 CAR T drugs. Both 28z and 28BBz ddPCR assays allow determination of the average vector copy number (VCN) per cell. We confirmed that the VCN is dependent on the multiplicity of infection and verified that the VCN of our experimental or GMP-like IL-1RAP CAR T cells met the requirement (< 5 VCN/cell) for delivery to the clinical department, similar to approved axi-cel or tisa-cel drugs. RESULTS: 28BBz and 28z ddPCR assays applied to 2 tumoral (acute myeloid leukemia (AML) or multiple myeloma (MM) xenograft humanized NSG mouse models allowed us to quantify the early expansion (up to day 30) of CAR T cells after injection. Interestingly, following initial expansion, when circulating CAR T cells were challenged with the tumor, we noted a second expansion phase. Investigation of the bone marrow, spleen and lung showed that CAR T cells disseminated more within these tissues in mice previously injected with leukemic cell lines. Finally, circulating CAR T cell ddPCR monitoring of R/R acute lymphoid leukemia or diffuse large B cell lymphoma (n = 10 for tisa-cel and n = 7 for axi-cel) patients treated with both approved CAR T cells allowed detection of early expansion, which was highly correlated with FC, as well as long-term persistence (up to 450 days), while FC failed to detect these events. CONCLUSION: Overall, we designed and validated 2 ddPCR assays allowing routine or preclinical monitoring of early- and long-term circulating approved or experimental CAR T cells, including our own IL-1RAP CAR T cells, which will be evaluated in an upcoming phase I clinical trial.


Assuntos
Variações do Número de Cópias de DNA , Linfoma Difuso de Grandes Células B , Animais , Antígenos CD19 , Xenoenxertos , Humanos , Imunoterapia Adotiva , Camundongos , Reação em Cadeia da Polimerase , Linfócitos T
3.
Mol Immunol ; 133: 154-162, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33667985

RESUMO

Identification of anti-human leukocyte antigen (HLA) antibodies (Abs) is based on Luminex™ technology. We used bioinformatics to (i) study the correlations of mean fluorescence intensities (MFIs) for all the possible allele pairs, and (ii) determine the degree of epitope homology between HLA antigens. Using MFI data on anti-HLA Abs from 6000 Luminex™ assays, we provide an updated overview of class I and II HLA antigen cross-reactivity in which each node corresponded to an allele and each link corresponded to a strong correlation between two alleles (Spearman's ρ > 0.8). We compared these correlations with the serological groups and the results of an epitope analysis. The strongest correlations concerned allele-specific Abs directed against the same antigen. For the HLA-A locus, the highest values of Spearman's ρ reflected broad specificity. For the HLA-B locus, graphs defined the HLA-Bw4 public epitope, and correlations between HLA-A and -B alleles were only present for beads with the same Bw4 public epitope. For the HLA-C locus, we identified two groups that differed with regard to their KIR ligand subclassification. Lastly, the HLA-DRB1 subgroups were part of a network. In the epitope analysis, Spearman's ρ was related to the number of matched epitopes within pairs of alleles. The combination of Spearman's ρ with simple, undirected graphing constitutes an effective tool for understanding routinely encountered cross-reactivity profiles. Based on this model, we have implemented an online data visualization tool available at http://cusureau.pythonanywhere.com/.


Assuntos
Especificidade de Anticorpos/imunologia , Epitopos/imunologia , Antígenos HLA/imunologia , Teste de Histocompatibilidade/métodos , Isoanticorpos/imunologia , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Humanos , Estudos Retrospectivos
5.
J Mol Diagn ; 23(4): 447-454, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33385585

RESUMO

Axicabtagene ciloleucel or axi-cel [CD19 chimeric antigen receptor (CAR) T cell] has been recently approved for refractory/relapsed diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma. Proliferation of CAR T cells after infusion and their persistence have been reported as important factors. Laboratory tools are needed for the monitoring of patients. We developed a vector-based, simple, and accurate real-time quantitative PCR (qPCR) to measure axi-cel vector copy number in peripheral blood samples. Primers and probe targeting the 5'LTR region of the gammaretroviral vector (mouse stem cell virus) were designed for amplification. To generate standard curves, mouse stem cell virus plasmid was subcultured and quantified using droplet digital PCR. The method was applied to quantify vector copy number in blood samples from patients treated with axi-cel. The limit of quantification of the qPCR assay was established at 2.2 copies/µL in DNA eluate. The qPCR method was well correlated with flow cytometry findings; however, the assay appeared to be more sensitive than flow cytometry. The kinetics observed in blood samples from treated patients were in agreement with previously reported findings. In conclusion, we developed a sensitive and accurate qPCR assay for the quantification of transgenic CAR T cells, which can be a useful additional tool for the monitoring of patients treated with axi-cel.

6.
HLA ; 97(2): 175-176, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33040464

RESUMO

HLA-DQB1*06:371 differs from HLA-DQB1*06:03:01:01 by one nucleotide substitution in codon 46 in exon 2.


Assuntos
Alelos , Cadeias beta de HLA-DQ , Éxons/genética , Cadeias beta de HLA-DQ/genética , Humanos , Análise de Sequência de DNA
7.
Cytometry B Clin Cytom ; 100(2): 218-224, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32841511

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is considered as a major scientific breakthrough in cancer immunotherapy. The success of adoptive CAR T-cell therapy for cancer has inspired researchers to expand indications into the area of solid tumors, autoimmune and infectious diseases. The most important factors influencing outcome and durability of the response after infusion of CAR T-cell are proliferation and persistence of this cell subset. It becomes therefore important to detect easily and monitor circulating CAR T-cells into blood samples. Approaches such as quantitative PCR (qPCR) or flow cytometry have been developed. The aim of this study was to set up and optimize a reachable flow cytometry technique using labeled CD19 protein for the measurement of CAR T-cells in infusion bag and patient's blood. METHODS: Patients receiving Yescarta in Cell Therapy Unit (Department of hematology, Lille university hospital, France) between April and October 2019 and healthy volunteers were included to set up the flow cytometry technique. RESULTS AND CONCLUSIONS: We assessed feasibility in clinic and suitability to routine workload of a flow cytometry technique to follow CAR T-cells in infusion bag and patient's blood. With only a few manual steps, the present protocol allows the technician to perform this technique among other routine tasks, meaning a time to results of <2 hr after sample reception. We were also able to assess CAR T-cell heterogenity in terms of CD4+ and CD8+ T lymphocytes within the subset. Moreover, this technique allows monitoring of both authority approved CD19 CAR T-cell.

8.
HLA ; 97(1): 82-83, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33040435

RESUMO

HLA-C*16:173 differs from HLA-C*16:01:01:01 by one nucleotide substitution in codon 36 in exon 2.


Assuntos
Genes MHC Classe I , Antígenos HLA-C , Alelos , Códon , Éxons/genética , Antígenos HLA-C/genética , Humanos , Análise de Sequência de DNA
9.
HLA ; 97(4): 382-383, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33350597

RESUMO

HLA-DQB1*06:374 differs from HLA-DQB1*06:02:01:01 by one nucleotide substitution in codon 62 in exon 2.


Assuntos
Alelos , Códon , Éxons/genética , Cadeias beta de HLA-DQ/genética , Humanos
10.
HLA ; 97(5): 436-437, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33350600

RESUMO

HLA-A*02:939 differs from HLA-A*02:01:01:01 by one nucleotide substitution in codon 222 in exon 4.


Assuntos
Antígenos HLA-A , Mutação de Sentido Incorreto , Alelos , Códon , Éxons/genética , Antígenos HLA-A/genética , Teste de Histocompatibilidade , Humanos , Análise de Sequência de DNA
11.
HLA ; 97(5): 447-448, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33369142

RESUMO

HLA-A*11:376 differs from HLA-A*11:01:01:01 by one nucleotide substitution in codon 168 in exon 3.


Assuntos
Antígenos HLA-A , Mutação de Sentido Incorreto , Alelos , Códon , Éxons/genética , Antígenos HLA-A/genética , Teste de Histocompatibilidade , Humanos , Análise de Sequência de DNA
12.
Clin Transl Immunology ; 9(12): e1217, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33376594

RESUMO

Objectives: Assessment of the adaptive immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for studying long-term immunity and vaccine strategies. We quantified IFNγ-secreting T cells reactive against the main viral SARS-CoV-2 antigens using a standardised enzyme-linked immunospot assay (ELISpot). Methods: Overlapping peptide pools built from the sequences of M, N and S viral proteins and a mix (MNS) were used as antigens. Using IFNγ T-CoV-Spot assay, we assessed T-cell and antibody responses in mild, moderate and severe SARS-CoV-2 patients and in control samples collected before the outbreak. Results: Specific T cells were assessed in 60 consecutive patients (mild, n = 26; moderate, n = 10; and severe patients, n = 24) during their follow-up (median time from symptom onset [interquartile range]: 36 days [28;53]). T cells against M, N and S peptide pools were detected in n = 60 (100%), n = 56 (93.3%), n = 55 patients (91.7%), respectively. Using the MNS mix, IFNγ T-CoV-Spot assay showed a specificity of 96.7% (95% CI, 88.5-99.6%) and a specificity of 90.3% (75.2-98.0%). The frequency of reactive T cells observed with M, S and MNS mix pools correlated with severity and with levels of anti-S1 and anti-RBD serum antibodies. Conclusion: IFNγ T-CoV-Spot assay is a reliable method to explore specific T cells in large cohorts of patients. This test may become a useful tool to assess the long-lived memory T-cell response after vaccination. Our study demonstrates that SARS-CoV-2 patients developing a severe disease achieve a higher adaptive immune response.

13.
HLA ; 96(3): 356-357, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32483903

RESUMO

HLA-DRB1*04:275 differs from HLA-DRB1*04:04:01 by one nucleotide substitution in codon 41 in exon 2.


Assuntos
Alelos , Sequência de Bases , Éxons/genética , Cadeias HLA-DRB1/genética , Humanos
14.
HLA ; 96(4): 513-514, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32506816

RESUMO

HLA-B*18:161 differs from HLA-B*18:01:01:01 by one nucleotide substitution in codon 136 in exon 3.


Assuntos
Genes MHC Classe I , Antígenos HLA-B , Alelos , Teste de Histocompatibilidade , Humanos , Análise de Sequência de DNA
15.
HLA ; 96(3): 369-370, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32506829

RESUMO

HLA-DQB1*02:141 differs from HLA-DQB1*02:02:01:01 by one nucleotide substitution at codon 55 in exon 2.


Assuntos
Alelos , Códon , Éxons/genética , Cadeias beta de HLA-DQ/genética , Humanos
16.
HLA ; 96(4): 544-545, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32543114

RESUMO

HLA-DQB1*03:01:46 differs from HLA-DQB1*03:01:01:01 by one nucleotide substitution in codon 142 in exon 3.


Assuntos
Alelos , Códon , Éxons/genética , Cadeias beta de HLA-DQ/genética , Humanos
17.
HLA ; 96(4): 515-516, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32519474

RESUMO

HLA-B*27:198 differs from HLA-B*27:05:02:01 by one nucleotide substitution in codon 45 in exon 2.


Assuntos
Genes MHC Classe I , Antígenos HLA-B , Alelos , Antígenos HLA-B/genética , Teste de Histocompatibilidade , Humanos , Análise de Sequência de DNA
18.
Bull Cancer ; 107(1S): S72-S84, 2020 Jan.
Artigo em Francês | MEDLINE | ID: mdl-31586527

RESUMO

Haploidentical hematopoietic stem cell transplantation has been growing steadily since 2012. The SFGM-TC has twice published guidelines concerning T-cell repleted haploidentical grafts with high dose cyclophosphamide post-transplantation. The 2013 workshop recommended using the non-myeloablative Baltimore protocol with bone marrow and developed prospective protocols to evaluate these transplantations. The 2015 workshop reported improved results of reduced conditioning regimens in Hodgkin's lymphoma and intensive conditioning in myeloid hemopathies, and a similar outcome with 10/10 HLA matched donor with the same disease-risk score thus raising the question of the qualifier "alternative" for haploidentical transplants. The current work concerns the criteria for selecting the donor. The main criterion remains the absence of anti-HLA antibodies directed against the donor present in the recipient sera (DSA - Donor Specific Antibodies). In case of DSA and in the absence of an alternative donor, desensitization protocols exist. The other criteria are impossible to prioritize: age, sex, CMV, and blood type. The degree of relatedness and the number of HLA incompatibilities do not seem to be a criterion of choice. The 'ideal' donor would be a young man, CMV-matched, without major ABO incompatibility with a marrow transplant. There is insufficient data for the KIR-ligand and NIMA/NIPA mismatch. Peripheral stem cell grafts appear to yield more acute GVHD than bone marrow grafts after intensive conditioning, but with comparable survival rates. Based on the literature review, the comparison of haploidentical with unrelated donors encourages inclusion in existing national protocols randomizing these different donors.


Assuntos
Haplótipos , Histocompatibilidade , Doadores de Tecidos , Adulto , Fatores Etários , Aloenxertos , Transplante de Medula Óssea , Ciclofosfamida/uso terapêutico , Infecções por Citomegalovirus/complicações , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Teste de Histocompatibilidade , Humanos , Imunossupressores/uso terapêutico , Depleção Linfocítica , Masculino , Fatores Sexuais , Linfócitos T , Condicionamento Pré-Transplante/efeitos adversos , Condicionamento Pré-Transplante/métodos
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