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1.
Anticancer Res ; 40(10): 5355-5359, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988854

RESUMO

BACKGROUND/AIM: Recent studies indicate that chimeric antigen receptor (CAR)-T-cells seem to be superior to CAR modified NK-92 cells. One, at least partial, explanation to this discrepancy has been addressed herein, by having NK-92 cells as target cells in cytotoxicity reactions using peripheral blood mononuclear cells. MATERIALS AND METHODS: A time-resolved fluorometric assay (TDA-labeled NK-92 or K562 as target cells) was used for measuring the cytotoxic activity of blood mononuclear cells (PBMC). RESULTS: The cytotoxic capacity of the NK-92 cells was initially demonstrated by their ability to efficiently kill K562 cells. Interestingly, having PBMC as effector cells rendered the very same NK-92 cells sensitive to NK-cell mediated cytolysis. A 1:100 target:effector ratio gave 34.1% lysis compared to 72.2% lysis for K562 cells. Incubating PBMC for longer times (24 up to 48 h) potentiated their NK-activity against NK-92 cells even more, reaching a level close to that obtained with K562 cells. CONCLUSION: This study pinpoints a severe problem that has to be considered in future immune-based cancer therapies with NK-92 as well as CAR-transduced NK-92 cells.


Assuntos
Imunoterapia Adotiva , Células Matadoras Naturais/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Neoplasias/terapia , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Células K562 , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia
2.
Nat Commun ; 11(1): 4166, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820173

RESUMO

T cells engineered to express chimeric antigen receptors (CAR-T cells) have shown impressive clinical efficacy in the treatment of B cell malignancies. However, the development of CAR-T cell therapies for solid tumors is hampered by the lack of truly tumor-specific antigens and poor control over T cell activity. Here we present an avidity-controlled CAR (AvidCAR) platform with inducible and logic control functions. The key is the combination of (i) an improved CAR design which enables controlled CAR dimerization and (ii) a significant reduction of antigen-binding affinities to introduce dependence on bivalent interaction, i.e. avidity. The potential and versatility of the AvidCAR platform is exemplified by designing ON-switch CARs, which can be regulated with a clinically applied drug, and AND-gate CARs specifically recognizing combinations of two antigens. Thus, we expect that AvidCARs will be a highly valuable platform for the development of controllable CAR therapies with improved tumor specificity.


Assuntos
Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo
4.
PLoS One ; 15(7): e0232915, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706785

RESUMO

Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell-impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.


Assuntos
Engenharia Genética/métodos , Regiões Promotoras Genéticas/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Lentivirus/genética , Células MCF-7 , RNA Mensageiro/genética , Transgenes/genética
5.
Proc Natl Acad Sci U S A ; 117(26): 14926-14935, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554495

RESUMO

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.


Assuntos
Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linhagem Celular , Citocinas/imunologia , Humanos , Lipocalinas/genética , Lipocalinas/imunologia , Conformação Molecular , Piperidinas/química , Piperidinas/farmacologia , Receptores de Antígenos Quiméricos/genética , Proteínas Plasmáticas de Ligação ao Retinol/genética , Proteínas Plasmáticas de Ligação ao Retinol/imunologia , Linfócitos T/efeitos dos fármacos
6.
Proc Natl Acad Sci U S A ; 117(26): 15148-15159, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541028

RESUMO

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.


Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos , Linhagem Celular Tumoral , Evolução Molecular Direcionada , Epitopos/genética , Humanos , Camundongos , Modelos Moleculares , Mutação , Conformação Proteica , Receptores de Antígenos Quiméricos/genética
7.
Anticancer Res ; 40(6): 3231-3237, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487617

RESUMO

BACKGROUND/AIM: We previously established a novel type of epidermal growth factor receptor variant III (EGFRvIII)-specific chimeric antigen receptor (CAR)-expressing natural killer (NK) cell line, designated EvCAR-KHYG-1, which inhibited the growth of glioblastoma (GBM) cells in vitro via apoptosis. MATERIALS AND METHODS: We investigated the cytokine-producing effect of EvCAR-KHYG-1 cells on GBM-like cell lines and their antitumour effect using in vivo xenograft assays. RESULTS: EvCAR-KHYG-1 cells produced interleukin-2, interferon-γ, and tumour necrosis factor-α on EGFRvIII-expressing U87MG cells. In vivo xenograft assays showed that EvCAR-KHYG-1 cells did not reduce the volume of subcutaneous tumours derived from EGFRvIII-expressing U87MG cells but did reduce tumour cell occupancy. CONCLUSION: EvCAR-KHYG-1 cells led to expression of cellular immunity-related cytokines on EGFRvIII-expressing U87MG in vitro but did not inhibit tumour progression due to the induction of a pseudo progression-like pathological feature. Future studies investigating the effect of different conditions in vivo are required to study the inhibition of tumour progression in GBM.


Assuntos
Neoplasias Encefálicas/terapia , Receptores ErbB/imunologia , Glioblastoma/terapia , Células Matadoras Naturais/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/imunologia , Progressão da Doença , Feminino , Glioblastoma/genética , Glioblastoma/imunologia , Glioblastoma/patologia , Células HEK293 , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição Aleatória , Receptores de Antígenos Quiméricos/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Biol Blood Marrow Transplant ; 26(7): 1239-1246, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32298807

RESUMO

The SARS-CoV-2 coronavirus (COVID-19) pandemic has significantly impacted the delivery of cellular therapeutics, including chimeric antigen receptor (CAR) T cells. This impact has extended beyond patient care to include logistics, administration, and distribution of increasingly limited health care resources. Based on the collective experience of the CAR T-cell Consortium investigators, we review and address several questions and concerns regarding cellular therapy administration in the setting of COVID-19 and make general recommendations to address these issues. Specifically, we address (1) necessary resources for safe administration of cell therapies; (2) determinants of cell therapy utilization; (3) selection among patients with B cell non-Hodgkin lymphomas and B cell acute lymphoblastic leukemia; (4) supportive measures during cell therapy administration; (5) use and prioritization of tocilizumab; and (6) collaborative care with referring physicians. These recommendations were carefully formulated with the understanding that resource allocation is of the utmost importance, and that the decision to proceed with CAR T cell therapy will require extensive discussion of potential risks and benefits. Although these recommendations are fluid, at this time it is our opinion that the COVID-19 pandemic should not serve as reason to defer CAR T cell therapy for patients truly in need of a potentially curative therapy.


Assuntos
Infecções por Coronavirus/epidemiologia , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Pandemias , Pneumonia Viral/epidemiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Linfócitos T/transplante , Anticorpos Monoclonais Humanizados/uso terapêutico , Controle de Doenças Transmissíveis , Infecções por Coronavirus/imunologia , Alocação de Recursos para a Atenção à Saúde/ética , Alocação de Recursos para a Atenção à Saúde/organização & administração , Humanos , Imunoterapia Adotiva/ética , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Pneumonia Viral/imunologia , Guias de Prática Clínica como Assunto , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Doadores de Tecidos/provisão & distribução , Estados Unidos/epidemiologia
9.
Nat Med ; 26(5): 712-719, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341579

RESUMO

Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. 3,4), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT5,6, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.


Assuntos
Antígenos B7/imunologia , Neoplasias Encefálicas/terapia , Vacinas Anticâncer/administração & dosagem , Imunoterapia Adotiva/métodos , Tumor Rabdoide/terapia , Teratoma/terapia , Adulto , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Encéfalo/patologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Células Cultivadas , Pré-Escolar , Feminino , Feto/patologia , Humanos , Lactente , Injeções Intraventriculares , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos Quiméricos/administração & dosagem , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Tumor Rabdoide/imunologia , Tumor Rabdoide/patologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Teratoma/imunologia , Teratoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Med ; 26(5): 720-731, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341580

RESUMO

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood-brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.


Assuntos
Neoplasias Encefálicas/terapia , Vacinas Anticâncer/administração & dosagem , Líquido Cefalorraquidiano/efeitos dos fármacos , Ependimoma/terapia , Imunoterapia Adotiva/métodos , Meduloblastoma/terapia , Animais , Neoplasias Encefálicas/líquido cefalorraquidiano , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Cerebelares/líquido cefalorraquidiano , Neoplasias Cerebelares/imunologia , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/terapia , Líquido Cefalorraquidiano/imunologia , Criança , Pré-Escolar , Sistemas de Liberação de Medicamentos/métodos , Ependimoma/líquido cefalorraquidiano , Ependimoma/imunologia , Ependimoma/patologia , Feminino , Células HEK293 , Humanos , Lactente , Injeções Intraventriculares , Masculino , Meduloblastoma/líquido cefalorraquidiano , Meduloblastoma/imunologia , Meduloblastoma/patologia , Camundongos , Metástase Neoplásica , Receptores de Antígenos Quiméricos/administração & dosagem , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Cell ; 37(4): 426-427, 2020 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-32289266

RESUMO

Chimeric antigen receptor (CAR)-T cell immunotherapy is highly effective against B lineage cancers but at the expense of potentially serious toxicity such as cytokine release syndrome (CRS) and neurotoxicity. Recent work provided clinical evidence that allogeneic, cord blood CAR-NK cells induce high rates of non-durable clinical responses without CRS or neurotoxicity.


Assuntos
Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Citocinas , Sangue Fetal , Células Matadoras Naturais , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos/genética
12.
J Vis Exp ; (157)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32250358

RESUMO

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.


Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Técnicas de Cultura de Células , Sobrevivência Celular , Imunoterapia Adotiva , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Transdução Genética
13.
Cancer Sci ; 111(5): 1478-1490, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32133731

RESUMO

The use of allogeneic, pluripotent stem-cell-derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator-initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) chimeric antigen receptor (CAR)-expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder-free production of CAR-expressing NK/ILC cells from CAR-transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8-3.6 × 106 iPSC within 7 weeks was 1.8-4.0 × 109 . These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN-γ) production against GPC3-expressing tumor cells. When the CAR-NK/ILC cells were injected into a GPC3-positive, ovarian-tumor-bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non-clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR-NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell-based immune cell cancer therapies.


Assuntos
Glipicanas/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Diferenciação Celular , Sobrevivência Celular , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Glipicanas/genética , Glipicanas/metabolismo , Humanos , Imunidade Inata , Imunoterapia Adotiva , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Interferon gama/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/transplante , Transfusão de Linfócitos , Linfócitos/citologia , Camundongos , Camundongos SCID , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo
14.
Int J Nanomedicine ; 15: 483-495, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158206

RESUMO

Background: The complex preparation procedures and severe toxicities are two major obstacles facing the wide use of chimeric antigen receptor-modified T (CAR-T) cells in clinical cancer immunotherapy. The nanotechnology-based T cell temporary CAR modification may be a potential approach to solve these problems and make the CAR-T cell-based tumor therapy feasible and broadly applicable. Methods: A series of plasmid DNA-loaded self-assembled nanoparticles (pDNA@SNPsx/y) prepared from adamantane-grafted polyamidoamine (Ad-PAMAM) dendrimers of different generations (G1 or G5) and cyclodextrin-grafted branched polyethylenimine (CD-PEI) of different molecular weights (800, 2000, or 25,000 Da) were characterized and evaluated. The detailed physicochemical properties, cellular interaction, and cytotoxicity of selected pDNA@SNPG1/800 were systematically investigated. Thereafter, the epidermal growth factor receptor variant III (EGFRvIII) CAR-expression plasmid vector (pEGFRvIII-CAR) was constructed and encapsulated into SNPG1/800. The resulting pEGFRvIII-CAR@SNPG1/800 was used for Jurkat cell transient transfection, and the EGFRvIII-CAR expressed in transfected cells was measured by flow cytometry and Western blot. Finally, the response of EGFRvIII CAR-positive Jurkat T cell to target tumor cell was evaluated. Results: The pDNA@SNPG1/800 showed the highest efficacy in Jurkat cell gene transfection and exhibited low cytotoxicity. pEGFRvIII-CAR@SNPG1/800 can efficiently deliver pEGFRvIII-CAR into Jurkat T cells, thereby resulting in transient EGFRvIII-CAR expression in transfected cells. EGFRvIII-CAR that is present on the cell membrane enabled Jurkat T cells to recognize and bind specifically with EGFRvIII-positive tumor cells. Conclusion: These results indicated that pEGFRvIII-CAR@SNPG1/800 can effectively achieve T-cell transient CAR modification, thereby demonstrating considerable potential in CAR-T cancer therapy.


Assuntos
Receptores ErbB/genética , Técnicas de Transferência de Genes , Imunoterapia Adotiva/métodos , Nanopartículas/química , Linfócitos T/fisiologia , Linhagem Celular Tumoral , Dendrímeros/química , Vetores Genéticos , Humanos , Iminas/química , Imunoterapia , Células Jurkat , Peso Molecular , Polietilenos/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/genética , Transfecção/métodos
15.
Sci Adv ; 6(8): eaay9209, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32128416

RESUMO

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.


Assuntos
Imunoterapia Adotiva , Luz , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/efeitos da radiação , Animais , Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Criptocromos/genética , Criptocromos/metabolismo , Citotoxicidade Imunológica/imunologia , Citotoxicidade Imunológica/efeitos da radiação , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Ativação Linfocitária/efeitos da radiação , Camundongos , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Receptores de Antígenos Quiméricos/genética , Ativação Transcricional/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Lancet Oncol ; 21(3): e168-e178, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32135120

RESUMO

The recent clinical successes of immunotherapy, as a result of a broader and more profound understanding of cancer immunobiology, and the leverage of this knowledge to effectively eradicate malignant cells, has revolutionised the field of cancer therapeutics. Immunotherapy is now considered the fifth pillar of cancer care, alongside surgery, chemotherapy, radiotherapy, and targeted therapy. Recently, the success of genetically modified T cells that express chimeric antigen receptors (CAR T cells) has generated considerable excitement. CAR T-cell therapy research and development has built on experience generated by laboratory research and clinical investigation of lymphokine-activated killer cells, tumour-infiltrating lymphocytes, and allogeneic haemopoietic stem-cell transplantation for cancer treatment. This Review aims to provide a background on the field of adoptive T-cell therapy and the development of genetically modified T cells, most notably CAR T-cell therapy. Many challenges exist to optimise efficacy, minimise toxicity, and broaden the application of immunotherapies based on T cells.


Assuntos
Imunoterapia Adotiva/tendências , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Animais , Difusão de Inovações , Previsões , Humanos , Imunoterapia Adotiva/efeitos adversos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Resultado do Tratamento , Microambiente Tumoral
18.
Transfusion ; 60(5): 1004-1014, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32167176

RESUMO

BACKGROUND: Most chimeric antigen receptor T (CAR-T) cells and other adoptive T-cell therapies (ACTs) are currently manufactured by ex vivo expansion of patient lymphocytes in culture media supplemented with human plasma from group AB donors. As lymphocytes do not express A or B antigens, the isoagglutinins of non-AB plasmas are unlikely to cause deleterious effects on lymphocytes in culture. STUDY DESIGN AND METHODS: Seeding cultures with peripheral blood mononuclear cell (PBMNC) concentrates from group A1 donors and using a CAR-T culture protocol, parallel cultures were performed, each with unique donor plasmas as media supplements (including group O plasmas with high-titer anti-A and group AB plasmas as control). An additional variable, a 3% group A1 red blood cell (RBC) spike, was added to simulate a RBC-contaminated PBMNC collection. Cultures were monitored by cell count, viability, flow cytometric phenotype, gene expression analysis, and supernatant chemokine analysis. RESULTS: There was no difference in lymphocyte expansion or phenotype when cultured with AB plasma or O plasma with high-titer anti-A. Compared to controls, the presence of contaminating RBCs in lymphocyte culture led to poor lymphocyte expansion and a less desirable phenotype-irrespective of the isoagglutinin titer of the plasma supplement used. CONCLUSIONS: This study suggests that ABO incompatible plasma may be used as a media supplement when culturing cell types that do not express ABO antigens-such as lymphocytes for CAR-T or other ACT. The presence of contaminating RBCs in culture was disadvantageous independent of isoagglutinin titer.


Assuntos
Sistema ABO de Grupos Sanguíneos/sangue , Meios de Cultura/química , Imunoterapia Adotiva , Plasma/fisiologia , Cultura Primária de Células/métodos , Linfócitos T/citologia , Células Cultivadas , Citometria de Fluxo , Antígenos HLA-A/sangue , Antígenos HLA-A/imunologia , Humanos , Imunoterapia Adotiva/métodos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Ativação Linfocitária , Plasma/química , Cultura Primária de Células/normas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Linfócitos T/transplante , Doadores de Tecidos
19.
Cell Mol Life Sci ; 77(18): 3567-3581, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32185403

RESUMO

Mammalian cells are inherently capable of sensing extracellular environmental signals and activating complex biological functions on demand. Advances in synthetic biology have made it possible to install additional capabilities, which can allow cells to sense the presence of custom biological molecules and provide defined outputs on demand. When implanted/infused in patients, such engineered cells can work as intrabody "doctors" that diagnose disease states and produce and deliver therapeutic molecules when and where necessary. The key to construction of such theranostic cells is the development of a range of sensor systems for detecting various extracellular environmental cues that can be rewired to custom outputs. In this review, we introduce the state-of-art engineering principles utilized in the design of sensor systems to detect soluble factors and also to detect specific cell contact, and we discuss their potential role in treating intractable diseases by delivering appropriate therapeutic functions on demand. We also discuss the challenges facing these emerging technologies.


Assuntos
Matriz Extracelular/metabolismo , Biologia Sintética/métodos , Animais , Técnicas Biossensoriais/métodos , Humanos , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Nanomedicina Teranóstica
20.
Nat Biotechnol ; 38(4): 448-459, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32015548

RESUMO

Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15.


Assuntos
Imunoterapia Adotiva/métodos , Interleucina-23/metabolismo , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-23/genética , Ativação Linfocitária , Camundongos , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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