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1.
Int J Cancer ; 138(5): 1256-68, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26376181

RESUMO

Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA(+) donor CD8(+) T cells with either single HLA antigen-mismatched or HLA-matched primary AML blasts, respectively, elicited strong leukemia reactivity during cytokine-optimized short to intermediate (i.e., 2-8 weeks) culture periods. Single doses of CTL were intravenously infused into NOD/scidIL2Rcg(null) mice when engraftment with patient AML reached bone marrow infiltration of 1-5%, clinically defining minimal residual disease status. This treatment resulted in complete regression of HLA-mismatched and strong reduction of HLA-matched AML infiltration, respectively. Most importantly, mice receiving AML-reactive CTL showed significantly prolonged survival. Transferred CTL were detectable in murine bone marrow and spleen and demonstrated sustained AML-reactivity ex vivo. Moreover, injections with human IL-15 clearly promoted CTL persistence. In summary, we show that naive donor-derived CD8(+) CTL effectively combat patient AML blasts in immunodeficient mice. The donor-patient specific humanized mouse model appears suitable to evaluate therapeutic efficacy of AML-reactive CTL before adoptive transfer into patients. It may further help to identify powerful leukemia rejection antigens and T-cell receptors for redirecting immunity to leukemias even in a patient-individualized manner.


Assuntos
Imunoterapia Adotiva , Leucemia Mieloide Aguda/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Efeito Enxerto vs Leucemia , Antígenos HLA-B/imunologia , Teste de Histocompatibilidade , Humanos , Leucemia Mieloide Aguda/imunologia , Camundongos , Neoplasia Residual , Medicina de Precisão
2.
Sci Transl Med ; 11(499)2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270272

RESUMO

Immunotherapy with chimeric antigen receptor (CAR)-engineered T cells can be effective against advanced malignancies. CAR T cells are "living drugs" that require technologies to enable physicians (and patients) to maintain control over the infused cell product. Here, we demonstrate that the tyrosine kinase inhibitor dasatinib interferes with the lymphocyte-specific protein tyrosine kinase (LCK) and thereby inhibits phosphorylation of CD3ζ and ζ-chain of T cell receptor-associated protein kinase 70 kDa (ZAP70), ablating signaling in CAR constructs containing either CD28_CD3ζ or 4-1BB_CD3ζ activation modules. As a consequence, dasatinib induces a function-off state in CD8+ and CD4+ CAR T cells that is of immediate onset and can be sustained for several days without affecting T cell viability. We show that treatment with dasatinib halts cytolytic activity, cytokine production, and proliferation of CAR T cells in vitro and in vivo. The dose of dasatinib can be titrated to achieve partial or complete inhibition of CAR T cell function. Upon discontinuation of dasatinib, the inhibitory effect is rapidly and completely reversed, and CAR T cells resume their antitumor function. The favorable pharmacodynamic attributes of dasatinib can be exploited to steer the activity of CAR T cells in "function-on-off-on" sequences in real time. In a mouse model of cytokine release syndrome (CRS), we demonstrated that a short treatment course of dasatinib, administered early after CAR T cell infusion, protects a proportion of mice from otherwise fatal CRS. Our data introduce dasatinib as a broadly applicable pharmacologic on/off switch for CAR T cells.


Assuntos
Dasatinibe/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Animais , Síndrome da Liberação de Citocina/imunologia , Dexametasona/farmacologia , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos SCID , Fosforilação/efeitos dos fármacos
3.
Nat Biotechnol ; 37(12): 1502-1512, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31685959

RESUMO

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (high-solubility SB, hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T cells, which exhibit potent antitumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.


Assuntos
Engenharia Genética/métodos , Mutagênese Insercional/genética , Transposases/genética , Engenharia Celular/métodos , Linhagem Celular , Células Cultivadas , Células HeLa , Humanos , Proteínas Recombinantes de Fusão/genética , Células-Tronco
4.
Adv Cell Gene Ther ; 1(2)2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30420973

RESUMO

OBJECTIVE: Integrins are heterodimeric receptors that convey cell-to-cell and cell-to-matrix interactions. Integrin αvß3 is expressed in several tumour entities including melanoma, glioblastoma, breast, pancreatic and prostate cancer, where it promotes tumour cell survival and metastasis. Here, we generated αvß3-specific chimeric antigen receptor (CAR) T-cells and analysed their antitumour function in pre-clinical models in vitro and in vivo. METHODS: αvß3-CARs comprising a super-humanised hLM609 targeting domain with either high or low affinity (hLM609v7, K d = 3 nM vs. hLM609v11, K d = 160 nM) and equipped with either a long or a short IgG4-Fc extracellular spacer (229 vs. 12 amino acids) were expressed in CD8+ and CD4+ T-cells through lentiviral transduction. RESULTS: αvß3-CAR T-cells eliminated αvß3-positive tumour cells rapidly and specifically, produced IFN-γ and IL-2 (CD4+ > CD8+) and exhibited productive proliferation. In vitro, we observed the strongest reactivity with the higher-affinity hLM609v7 αvß3-CAR in the short spacer configuration, consistent with the tumour membrane-distal localization of the hLM609 epitope. In a murine xenograft model of metastatic A-375 melanoma, the strongest antitumour effect was mediated by the lower-affinity hLM609v11 αvß3-CAR. Notably, a single administration of hLM609v11 αvß3-CAR T-cells was able to induce complete elimination of melanoma lesions, leading to long-term tumour-free survival. CONCLUSIONS: These data establish αvß3 integrin as a novel target for CAR T-cell immunotherapy, and affirm our previous notion that binding domain affinity and spacer length can be calibrated to augment CAR reactivity. CLINICAL IMPLICATIONS: αvß3-CAR T-cells have therapeutic potential in several prevalent solid tumours, including melanoma and triple-negative breast cancer.

5.
PLoS One ; 7(11): e49243, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23166619

RESUMO

The Sec61 translocon of the endoplasmic reticulum (ER) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. Protein translocation into the ER can occur co- and posttranslationally. In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning ER reporters are not affected. Consistent with this, the knock-down of Sec63 increases the steady-state pools of polytopic ER proteins, suggesting a substrate-specific and regulatory function of Sec63 in ER import. Overexpressed Sec63 exerts its down-regulating activity on polytopic protein levels independent of its Sec62-interacting motif, indicating that it may not act in conjunction with Sec62 in human cells. The specific action of Sec63 is further sustained by our observations that the up-regulation of either Sec62 or two other ER proteins with lumenal J domains, like ERdj1 and ERdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. A J domain-specific mutation of Sec63, proposed to weaken its interaction with the ER resident BiP chaperone, reduces the down-regulating capacity of excess Sec63, suggesting an involvement of BiP in this process. Together, these results suggest that Sec63 may perform a substrate-selective quantity control function during cotranslational ER import.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Bactérias , Western Blotting , Linhagem Celular , Primers do DNA/genética , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Luminescentes , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência , Chaperonas Moleculares , Mutação/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas do Envelope Viral/metabolismo
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