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1.
Gene Ther ; 30(9): 706-713, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37173386

RESUMO

Even if adoptive cell transfer (ACT) has already shown great clinical efficiency in different types of disease, such as cancer, some adverse events consistently occur, and suicide genes are an interesting system to manage these events. Our team developed a new medical drug candidate, a chimeric antigen receptor (CAR) targeting interleukin-1 receptor accessory protein (IL-1RAP), which needs to be evaluated in clinical trials with a clinically applicable suicide gene system. To prevent side effects and ensure the safety of our candidate, we devised two constructs carrying an inducible suicide gene, RapaCasp9-G or RapaCasp9-A, containing a single-nucleotide polymorphism (rs1052576) affecting the efficiency of endogenous caspase 9. These suicide genes are activated by rapamycin and based on the fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization. RapaCasp9-G- and RapaCasp9-A-expressing gene-modified T cells (GMTCs) were produced from healthy donors (HDs) and acute myeloid leukemia (AML) donors. The RapaCasp9-G suicide gene demonstrated better efficiency, and we showed its in vitro functionality in different clinically relevant culture conditions. Moreover, as rapamycin is not pharmacologically inert, we also demonstrated its safe use as part of our therapy.


Assuntos
Imunoterapia Adotiva , Proteína Acessória do Receptor de Interleucina-1 , Humanos , Caspase 9/genética , Caspase 9/metabolismo , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Imunoterapia Adotiva/efeitos adversos , Linfócitos T , Sirolimo/metabolismo
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.
Curr Res Transl Med ; 71(2): 103385, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36773434

RESUMO

PURPOSE OF THE STUDY: The use of chimeric antigen receptor (CAR)-T cells has demonstrated excellent results in B-lymphoid malignancies. The Advanced Therapy Medicinal Products (ATMP) status and good manufacturing practice (GMP) of CAR-T cells require particular conditions of production performed in a pharmaceutical establishment. Our team developed a new medical drug candidate for acute myeloid leukemia (AML), a CAR targeting interleukin-1 receptor accessory protein (IL-1RAP) expressed by leukemia stem cells, which will need to be evaluated in a phase I-IIa clinical trial. During the preclinical development phase, we produced IL-1RAP CAR-T cells in a semi-automated closed system (CliniMACSࣨ Prodigy) using research grade lentiviral particles. PATIENTS AND THE METHODS: The purpose of this work was to validate our production process and to characterize our preclinical GMP-like medicinal product. IL-1RAP CAR-T cells were produced from healthy donors in 9 days, either in an semi-automated closed system (with GMP-like compliant conditions) or according to another research protocols, which was used as a reference. RESULTS: Based on phenotypic, functional and metabolic analyses, we were able to show that the final product is ready for clinical use. Finally, in a xenograft AML murine model, we demonstrated that the IL-1RAP CAR-T cells generated in a GMP-like environment could eliminate tumor cells and increase overall survival. CONCLUSION: We demonstrated that our IL-1RAP CAR-T cell preclinical GMP-like production process meets standard regulatory requirements in terms of CAR-T cell number, subpopulation phenotype and cytotoxic functionality. Our CAR-T cell production process was validated and can be used to produce medicinal IL-1RAP CAR-T cells for the first phase I clinical trial.


Assuntos
Imunoterapia Adotiva , Proteína Acessória do Receptor de Interleucina-1 , Humanos , Animais , Camundongos , Imunoterapia Adotiva/métodos , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Linfócitos T/metabolismo , Fenótipo
4.
J Immunother Cancer ; 10(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35803613

RESUMO

BACKGROUND: Acute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are the basis of refractory/relapsed (R/R) disease in 80% of patients with AML not receiving allogeneic transplantation. METHODS: In this study, we showed that the interleukin-1 receptor accessory protein (IL-1RAP) protein is overexpressed on the cell surface of LSCs in all subtypes of AML and confirmed it as an interesting and promising target in AML compared with the most common potential AML targets, since it is not expressed by the normal hematopoietic stem cell. After establishing the proof of concept for the efficacy of chimeric antigen receptor (CAR) T-cells targeting IL-1RAP in chronic myeloid leukemia, we hypothesized that third-generation IL-1RAP CAR T-cells could eliminate AML LSCs, where the medical need is not covered. RESULTS: We first demonstrated that IL-1RAP CAR T-cells can be produced from AML T-cells at the time of diagnosis and at relapse. In vitro and in vivo, we showed the effectiveness of IL-1RAP CAR T-cells against AML cell lines expressing different levels of IL-1RAP and the cytotoxicity of autologous IL-1RAP CAR T-cells against primary cells from patients with AML at diagnosis or at relapse. In patient-derived relapsed AML xenograft models, we confirmed that IL-1RAP CAR T-cells are able to circulate in peripheral blood and to migrate in the bone marrow and spleen, are cytotoxic against primary AML cells and increased overall survival. CONCLUSION: In conclusion, our preclinical results suggest that IL-1RAP CAR T-based adoptive therapy could be a promising strategy in AML treatment and it warrants the clinical investigation of this CAR T-cell therapy.


Assuntos
Leucemia Mieloide Aguda , Receptores de Antígenos Quiméricos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imunoterapia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Leucemia Mieloide Aguda/terapia , Recidiva , Linfócitos T
5.
Cancer Gene Ther ; 28(12): 1365-1375, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33414517

RESUMO

Although chimeric antigen receptor CAR) T cell immunotherapies are an undeniable and unequivocal success, knowledge obtained from the monitoring of the first clinical trials targeting the CD19 antigen in B malignancies, either refractory/relapsed acute lymphoid leukemia (ALL) or lymphomas, contributed to the identification of tumor cell escape in about 30-50% of B-ALL. Resistance occurred due to loss of surface expression of the antigen (rCD19-) or to the early disappearance or inactivation of CAR T cells (rCD19+). In a recently reported clinical case, rCD19- relapse resulted from masking of the antigen by the CAR at the surface of B-ALL leukemia cells following the unexpected viral transduction of a leukemic cell present in the cytapheresis sample. The objective of this work was to reproduce this epitope-masking resistance model, in the context of acute myeloid leukemia (AML), based on our immunotherapeutic CAR T cell model targeting the accessory protein of the interleukin-1 receptor (IL-1RAP) expressed by leukemic stem cells. As AML primary blasts express different levels of IL-1RAP, we modeled transduction of different AML tumor cell lines screened for density of antigenic sites with our lentiviral vectors carrying a third-generation IL-1RAP CAR, an iCASP9 suicide gene, and a truncated CD19 surface gene. We demonstrated that primary AML blasts can be easily transduced (74.55 ± 21.29%, n = 4) and that CAR T cytotoxicity to IL-1RAP is inversely correlated with epitope masking in relation to the number of antigenic sites expressed on the surface of IL-1RAP+ lines. Importantly, we showed that, in vitro, a 24-h exposure of IL-1RAP+/CAR+ leukemia lines to Rimiducid eliminated >85% of the cells. We confirmed that the expression of IL-1RAP CAR by an IL-1RAP+ leukemic cell, by decreasing the membrane availability of the targeted antigen, can induce resistance while a high epitope density maintains sensitivity to CAR T cells. Moreover, the presence of the iCASP9/Rimiducid suicide system safety switch makes this immunotherapy approach safe for application in a future phase 1 clinical trial.


Assuntos
Caspase 9/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Epitopos/metabolismo , Imunoterapia/métodos , Leucemia Mieloide Aguda/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Med Sci (Paris) ; 35(6-7): 497-500, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31274074

Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Hematológicas/terapia , Imunoterapia Adotiva/métodos , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Anticorpos Monoclonais/genética , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Caspase 9/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Transgênicos Suicidas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoterapia Adotiva/efeitos adversos , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplante , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos Quiméricos/imunologia
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