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1.
Nature ; 604(7906): 563-570, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35418687

RESUMO

Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.


Assuntos
Glioblastoma , Receptores de Antígenos Quiméricos , Morte Celular , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Imunoterapia Adotiva , Linfócitos T/patologia
2.
Blood ; 144(11): 1153-1167, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-38781564

RESUMO

ABSTRACT: We report a first-in-human clinical trial using chimeric antigen receptor (CAR) T cells targeting CD37, an antigen highly expressed in B- and T-cell malignancies. Five patients with relapsed or refractory CD37+ lymphoid malignancies were enrolled and infused with autologous CAR-37 T cells. CAR-37 T cells expanded in the peripheral blood of all patients and, at peak, comprised >94% of the total lymphocytes in 4 of 5 patients. Tumor responses were observed in 4 of 5 patients with 3 complete responses, 1 mixed response, and 1 patient whose disease progressed rapidly and with relative loss of CD37 expression. Three patients experienced prolonged and severe pancytopenia, and in 2 of these patients, efforts to ablate CAR-37 T cells, which were engineered to coexpress truncated epidermal growth factor receptor, with cetuximab were unsuccessful. Hematopoiesis was restored in these 2 patients after allogeneic hematopoietic stem cell transplantation. No other severe, nonhematopoietic toxicities occurred. We investigated the mechanisms of profound pancytopenia and did not observe activation of CAR-37 T cells in response to hematopoietic stem cells in vitro or hematotoxicity in humanized models. Patients with pancytopenia had sustained high levels of interleukin-18 (IL-18) with low levels of IL-18 binding protein in their peripheral blood. IL-18 levels were significantly higher in CAR-37-treated patients than in both cytopenic and noncytopenic cohorts of CAR-19-treated patients. In conclusion, CAR-37 T cells exhibited antitumor activity, with significant CAR expansion and cytokine production. CAR-37 T cells may be an effective therapy in hematologic malignancies as a bridge to hematopoietic stem cell transplant. This trial was registered at www.ClinicalTrials.gov as #NCT04136275.


Assuntos
Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Masculino , Pessoa de Meia-Idade , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/efeitos adversos , Feminino , Receptores de Antígenos Quiméricos/imunologia , Adulto , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos CD , Idoso , Antígenos de Neoplasias/imunologia , Antígenos CD7/metabolismo , Transplante de Células-Tronco Hematopoéticas , Recidiva , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Tetraspaninas
3.
Annu Rev Med ; 72: 365-382, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-32776808

RESUMO

Adoptive transfer of T cells modified with chimeric antigen receptors (CAR-T cells) has changed the therapeutic landscape of hematological malignancies, particularly for acute lymphoblastic leukemia and large B cell lymphoma, where two different CAR-T products are now considered standard of care. Furthermore, intense research efforts are under way to expand the clinical application of CAR-T cell therapy for the benefit of patients suffering from other types of cancers. Nevertheless, CAR-T cell treatment is associated with toxicities such as cytokine release syndrome, which can range in severity from mild flu-like symptoms to life-threatening vasodilatory shock, and a neurological syndrome termed ICANS (immune effector cell-associated neurotoxicity syndrome), which can also range in severity from a temporary cognitive deficit lasting only a few hours to lethal cerebral edema. In this review, we provide an in-depth discussion of different types of CAR-T cell-associated toxicities, including an overview of clinical presentation and grading, pathophysiology, and treatment options. We also address future perspectives and opportunities, with a special focus on hematological malignancies.


Assuntos
Neoplasias Hematológicas/terapia , Imunidade Celular , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Neoplasias Hematológicas/imunologia , Humanos
5.
Cell Mol Life Sci ; 76(2): 369-380, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30357422

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously regenerate the hematopoietic system through differentiation and self-renewal. The process of differentiation is initiated in the G1 phase of the cell cycle, when stem cells leave their quiescent state. During G1, the anaphase-promoting complex or cyclosome associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate cell proliferation. Following Cdh1 knockdown in HSPCs, we analyzed human and mouse hematopoiesis in vitro and in vivo in competitive transplantation assays. We found that Cdh1 is highly expressed in human CD34+ HSPCs and downregulated in differentiated subsets; whereas, loss of Cdh1 restricts myeloid differentiation, supports B cell development and preserves immature short-term HSPCs without affecting proliferation or viability. Our data highlight a role of Cdh1 as a regulator of balancing the maintenance of HSPCs and differentiation into mature blood cells.


Assuntos
Proteínas Cdh1/metabolismo , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas Cdh1/antagonistas & inibidores , Proteínas Cdh1/genética , Proliferação de Células , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/deficiência , Proteínas Proto-Oncogênicas c-kit/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
6.
Clin Exp Nephrol ; 23(2): 199-206, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30128942

RESUMO

BACKGROUND: The estimated glomerular filtration rate (eGFR) is clinically used to approximate renal function and adapt drug dosage. Multiple myeloma is a hematological disease; its prognosis is largely influenced by renal function. We evaluated two commonly used GFR estimations, CKD-EPI and MDRD (CKD Epidemiology Collaboration; Modification of Diet in Renal Disease) in myeloma patients undergoing treatment with lenalidomide, a renally excreted immunomodulatory drug. METHODS: We prospectively studied 130 myeloma patients receiving lenalidomide treatment at our institution. At baseline and after 3 months, GFR estimations were performed based on the CKD-EPI and MDRD equations. We compared eGFR-dependent CKD staging and lenalidomide dosage assignments. RESULTS: Initially, most patients were classified as CKD stage I/II, using both equations. Comparison of baseline renal function via CKD-EPI and MDRD induced concordance of CKD staging in 83% of patients, while CKD-EPI improved CKD staging in 16% of patients (p = 0.11). CKD-EPI assigned 3% of patients to higher lenalidomide dosing as opposed to MDRD. Both equations showed improved eGFR after 3 months of lenalidomide treatment. CONCLUSIONS: In our multiple myeloma patient cohort, CKD-EPI and MDRD led to similar CKD staging with minor differences in lenalidomide dosage assignment. Consistent with previous studies, eGFR improved under lenalidomide treatment. To standardize GFR estimation in myeloma patients, we suggest using the CKD-EPI equation.


Assuntos
Antineoplásicos/administração & dosagem , Taxa de Filtração Glomerular , Rim/fisiopatologia , Lenalidomida/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Insuficiência Renal Crônica/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Creatinina/sangue , Cálculos da Dosagem de Medicamento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/diagnóstico , Estudos Prospectivos , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/fisiopatologia , Fatores de Tempo , Resultado do Tratamento
7.
Clin Cancer Res ; 30(9): 1859-1877, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38393682

RESUMO

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.


Assuntos
Complexo CD3 , Endopeptidases , Proteínas Ligadas por GPI , Imunoterapia Adotiva , Mesotelina , Neoplasias Pancreáticas , Receptores de Antígenos Quiméricos , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Camundongos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Linhagem Celular Tumoral , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/terapia , Adenocarcinoma/patologia
8.
Haematologica ; 98(12): 1886-95, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23812934

RESUMO

Spindle poison-based therapy is of only limited benefit in acute myeloid leukemia while lymphoblastic leukemia/lymphoma responds well. In this study, we demonstrated that the spindle assembly checkpoint protein BubR1 was down-regulated in the vast majority of cases of acute myeloid leukemia whereas its expression was high in lymphoblastic cells. Correct function of the spindle assembly checkpoint is pivotal in mediating mitotic delay in response to spindle poisons. Mitotic delay by the spindle assembly checkpoint is achieved by inhibition of anaphase-promoting complex-dependent proteolysis of cyclin B and securin. We demonstrated a link between the repression of the spindle assembly checkpoint protein BubR1 in acute myeloid leukemia and the limited response to spindle poison. In accordance with its established role as an anaphase-promoting complex-inhibitor, we found that repression of BubR1 was associated with enhanced anaphase-promoting complex activity and cyclin B and securin degradation, which leads to premature sister-chromatid separation and failure to sustain a mitotic arrest. This suggests that repression of BubR1 in acute myeloid leukemia renders the spindle assembly checkpoint-mediated inhibition of the anaphase-promoting complex insufficient, which facilitates completion of mitosis in the presence of spindle poison. As both direct and BubR1-mediated restoration of cyclin B expression enhanced response to spindle poison, we propose that the downstream axis of the spindle assembly checkpoint is a promising target for tailored therapies for acute myeloid leukemia.


Assuntos
Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Células HL-60 , Células HeLa , Humanos , Leucemia Mieloide Aguda/genética , Mitose/genética , Dados de Sequência Molecular , Nocodazol/farmacologia , Nocodazol/uso terapêutico , Proteínas Serina-Treonina Quinases/genética
9.
Nat Commun ; 14(1): 7509, 2023 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-37980341

RESUMO

Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting.


Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Mieloma Múltiplo/metabolismo , Imunoterapia Adotiva , Antígeno de Maturação de Linfócitos B/genética , Linfócitos T
10.
Neurooncol Adv ; 5(1): vdac185, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36751672

RESUMO

Background: Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogeneous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape. Methods: Here we have developed a novel, dual-specific, tandem CAR T (TanCART) cell with the ability to simultaneously target both EGFRvIII and IL-13Rα2, two well-characterized tumor antigens that are frequently found on the surface of GBM cells but completely absent from normal brain tissues. We employed both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogeneous tumors. Results: Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < .05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete, and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < .05). Conclusions: We demonstrate that TanCART is effective against heterogeneous tumors in the brain. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.

11.
Nat Biotechnol ; 40(2): 189-193, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-33927418

RESUMO

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Peixe-Zebra , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/genética , Ribonucleoproteínas/genética , Peixe-Zebra/genética
12.
Cancer Cell ; 40(5): 494-508.e5, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35452603

RESUMO

Chimeric antigen receptor (CAR) T cell therapy is effective in lymphoid malignancies, but there has been limited data in myeloid cancers. Here, we start with a CD27-based CAR to target CD70 ("native") in acute myeloid leukemia (AML), and we find modest efficacy in vivo, consistent with prior reports. We then use orthogonal approaches to increase binding on both the tumor and CAR-T cell sides of the immune synapse: a pharmacologic approach (azacitidine) to increase antigen density of CD70 in myeloid tumors, and an engineering approach to stabilize binding of the CAR to CD70. To accomplish the latter, we design a panel of hinge-modified regions to mitigate cleavage of the extracellular portion of CD27. Our CD8 hinge and transmembrane-modified CD70 CAR-T cells are less prone to cleavage, have enhanced binding avidity, and increased expansion, leading to more potent in vivo activity. This enhanced CD70-targeted CAR is a promising candidate for further clinical development.


Assuntos
Leucemia Mieloide Aguda , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva , Leucemia Mieloide Aguda/terapia , Linfócitos T
13.
Blood Cancer Discov ; 3(2): 136-153, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35015685

RESUMO

Chimeric antigen receptor (CAR) T cells induce impressive responses in patients with hematologic malignancies but can also trigger cytokine release syndrome (CRS), a systemic toxicity caused by activated CAR T cells and innate immune cells. Although IFNγ production serves as a potency assay for CAR T cells, its biologic role in conferring responses in hematologic malignancies is not established. Here we show that pharmacologic blockade or genetic knockout of IFNγ reduced immune checkpoint protein expression with no detrimental effect on antitumor efficacy against hematologic malignancies in vitro or in vivo. Furthermore, IFNγ blockade reduced macrophage activation to a greater extent than currently used cytokine antagonists in immune cells from healthy donors and serum from patients with CAR T-cell-treated lymphoma who developed CRS. Collectively, these data show that IFNγ is not required for CAR T-cell efficacy against hematologic malignancies, and blocking IFNγ could simultaneously mitigate cytokine-related toxicities while preserving persistence and antitumor efficacy. SIGNIFICANCE: Blocking IFNγ in CAR T cells does not impair their cytotoxicity against hematologic tumor cells and paradoxically enhances their proliferation and reduces macrophage-mediated cytokines and chemokines associated with CRS. These findings suggest that IFNγ blockade may improve CAR T-cell function while reducing treatment-related toxicity in hematologic malignancies. See related content by McNerney et al., p. 90 (17). This article is highlighted in the In This Issue feature, p. 85.


Assuntos
Neoplasias Hematológicas , Imunoterapia Adotiva , Síndrome da Liberação de Citocina , Citocinas/metabolismo , Neoplasias Hematológicas/terapia , Humanos , Imunoterapia Adotiva/efeitos adversos , Interferon gama/metabolismo , Ativação de Macrófagos , Linfócitos T/metabolismo
14.
Sci Transl Med ; 13(575)2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33408186

RESUMO

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.


Assuntos
Lenalidomida , Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Células Jurkat , Receptores de Antígenos de Linfócitos T , Ubiquitina-Proteína Ligases
15.
J Immunother Cancer ; 8(2)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32900862

RESUMO

BACKGROUND: Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure. METHODS: We created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies. RESULTS: We found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma. CONCLUSIONS: Our aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Imunoterapia Adotiva/métodos , Lentivirus/genética , Transdução Genética/métodos , Humanos
16.
Adv Sci (Weinh) ; 6(23): 1901829, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31832320

RESUMO

Cancer immunotherapy based on the engineering of chimeric antigen receptors (CAR) on T cells has emerged as one of the most promising new therapies for patients with B-cell malignancies. Preclinical assessments of essential CAR T cell functions such as trafficking and cytotoxicity are critical for accelerating the development of highly effective therapeutic candidates. However, current tools for evaluating CAR-T functions lack sufficient precision. Here, a micropatterned tumor array (MiTA) is described that enables detailed and dynamic characterization of CAR T cell trafficking toward tumor-cell islands and subsequent killing of tumor cells. It is shown that CAR T cells often merge into large clusters that envelop and kill the tumor cells with high efficiency. Significant differences are also measured between CAR T cells from different donors and between various CAR T cell constructs. Overall, the assay allows for multifaceted, dynamic, high-content evaluation of CAR T trafficking, clustering, and killing and could eventually become a useful tool for immune-oncology research and preclinical assessments of cell-based immunotherapies.

17.
Nat Biotechnol ; 37(9): 1049-1058, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31332324

RESUMO

Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antígenos de Neoplasias/imunologia , Glioblastoma/terapia , Receptores de Antígenos Quiméricos , Animais , Antígenos de Neoplasias/metabolismo , Diferenciação Celular , Receptores ErbB , Glioblastoma/imunologia , Glioblastoma/metabolismo , Humanos , Camundongos , Neoplasias Experimentais , Linfócitos T/fisiologia
18.
Blood Adv ; 3(21): 3248-3260, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31698455

RESUMO

Chimeric antigen receptor (CAR) T cells (CARTs) have shown tremendous potential for the treatment of certain B-cell malignancies, including patients with relapsed/refractory multiple myeloma (MM). Targeting the B-cell maturation antigen (BCMA) has produced the most promising results for CART therapy of MM to date, but not all remissions are sustained. Emergence of BCMA escape variants has been reported under the selective pressure of monospecific anti-BCMA CART treatment. Thus, there is a clinical need for continuous improvement of CART therapies for MM. Here, we show that a novel trimeric APRIL (a proliferation-inducing ligand)-based CAR efficiently targets both BCMA+ and BCMA- MM. Modeled after the natural ligand-receptor pair, APRIL-based CARs allow for bispecific targeting of the MM-associated antigens BCMA and transmembrane activator and CAML interactor (TACI). However, natural ligands as CAR antigen-binding domains may require further engineering to promote optimal binding and multimerization to adequately trigger T-cell activation. We found that using a trimeric rather than a monomeric APRIL format as the antigen-binding domain enhanced binding to BCMA and TACI and CART activity against MM in vitro and in vivo. Dual-specific, trimeric APRIL-based CAR are a promising therapeutic approach for MM with potential for preventing and treating BCMA escape.


Assuntos
Antígenos de Neoplasias , Imunoterapia Adotiva , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Antígenos de Neoplasias/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Camundongos , Ligação Proteica/imunologia , Receptores de Antígenos Quiméricos/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Immunother Cancer ; 7(1): 304, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727131

RESUMO

Despite remarkable success in the treatment of hematological malignancies, CAR T-cell therapies for solid tumors have floundered, in large part due to local immune suppression and the effects of prolonged stimulation leading to T-cell dysfunction and exhaustion. One mechanism by which gliomas and other cancers can hamper CAR T cells is through surface expression of inhibitory ligands such as programmed cell death ligand 1 (PD-L1). Using the CRIPSR-Cas9 system, we created universal CAR T cells resistant to PD-1 inhibition through multiplexed gene disruption of endogenous T-cell receptor (TRAC), beta-2 microglobulin (B2M) and PD-1 (PDCD1). Triple gene-edited CAR T cells demonstrated enhanced activity in preclinical glioma models. Prolonged survival in mice bearing intracranial tumors was achieved after intracerebral, but not intravenous administration. CRISPR-Cas9 gene-editing not only provides a potential source of allogeneic, universal donor cells, but also enables simultaneous disruption of checkpoint signaling that otherwise impedes maximal antitumor functionality.


Assuntos
Neoplasias Encefálicas/terapia , Receptores ErbB , Glioblastoma/terapia , Imunoterapia Adotiva , Receptor de Morte Celular Programada 1/genética , Animais , Neoplasias Encefálicas/imunologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Glioblastoma/imunologia , Humanos , Camundongos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Clin Cancer Res ; 25(23): 7046-7057, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31439577

RESUMO

PURPOSE: T cells engineered to express a chimeric antigen receptor (CAR) against CD19 have recently been FDA approved for the treatment of relapsed or refractory large B-cell lymphoma. Despite the success and curative potential of CD19 CAR T cells, several reports describing disease relapse due to antigen loss are now emerging. EXPERIMENTAL DESIGN: We developed a novel CAR construct directed against CD79b, a critical receptor for successful B-cell development that remains highly expressed in several subtypes of B-cell lymphoma, including mantle cell lymphoma (MCL). We tested CAR T cells directed against CD79b alone or in combination with CD19 targeting in a single construct, against cell line- and patient-derived xenograft models. RESULTS: We demonstrate CAR79b antigen-specific recognition and cytotoxicity against a panel of cell lines and patient-derived xenograft models of MCL. Importantly, we show that downregulation of CD19 does not influence surface expression of CD79b and that anti-CD79b CAR T cells alone or arranged in a dual-targeting format with a CD19 single-chain variable fragment (scFv) are able to recognize and eliminate CD19+, CD19-, and mixed CD19+/CD19-B-cell lymphoma. CONCLUSIONS: Our findings demonstrate that CAR T cells targeting CD79b alone or in combination have promise for treating and preventing CD19 antigen escape in B-cell lymphomas.


Assuntos
Antígenos CD19/imunologia , Antígenos CD79/imunologia , Imunoterapia Adotiva/métodos , Linfoma de Célula do Manto/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Apoptose , Proliferação de Células , Humanos , Ativação Linfocitária , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
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