RESUMEN
Patients with small cell lung cancer (SCLC) generally have a poor prognosis and a median overall survival of only about 13 months, indicating the urgent need for novel therapies. Delta-like protein 3 (DLL3) has been identified as a tumor-specific cell surface marker on neuroendocrine cancers, including SCLC. In this study, we developed a chimeric antigen receptor (CAR) against DLL3 that displays antitumor efficacy in xenograft and murine SCLC models. CAR T cell expression of the proinflammatory cytokine IL-18 greatly enhanced the potency of DLL3-targeting CAR T cell therapy. In a murine metastatic SCLC model, IL-18 production increased the activation of both CAR T cells and endogenous tumor-infiltrating lymphocytes. We also observed an increased infiltration, repolarization, and activation of antigen-presenting cells. Additionally, human IL-18-secreting anti-DLL3 CAR T cells showed an increased memory phenotype, less exhaustion, and induced durable responses in multiple SCLC models, an effect that could be further enhanced with anti-PD-1 blockade. All together, these results define DLL3-targeting CAR T cells that produce IL-18 as a potentially promising novel strategy against DLL3-expressing solid tumors.
Asunto(s)
Carcinoma Neuroendocrino , Interleucina-18 , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Animales , Humanos , Ratones , Línea Celular Tumoral , Inmunoterapia Adoptiva , Interleucina-18/farmacología , Interleucina-18/uso terapéutico , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Linfocitos T/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable responses in B-cell malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR T cells and immune escape. These clinical challenges have compromised the long-term efficacy of CAR T-cell therapy and call for the development of novel CAR designs. We demonstrated that CAR T cells secreting a cytokine interleukin-36γ (IL-36γ) showed significantly improved CAR T-cell expansion and persistence, and resulted in superior tumor eradication compared with conventional CAR T cells. The enhanced cellular function by IL-36γ was mediated through an autocrine manner. In addition, activation of endogenous antigen-presenting cells (APCs) and T cells by IL-36γ aided the formation of a secondary antitumor response, which delayed the progression of antigen-negative tumor challenge. Together, our data provide preclinical evidence to support the translation of this design for an improved CAR T-cell-mediated antitumor response.
Asunto(s)
Inmunidad Celular/inmunología , Inmunoterapia Adoptiva/métodos , Interleucina-1/metabolismo , Activación de Linfocitos/inmunología , Linfoma de Células T/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Células Presentadoras de Antígenos , Apoptosis , Proliferación Celular , Femenino , Humanos , Interleucina-1/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Linfoma de Células T/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Factor 88 de Diferenciación Mieloide/fisiología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3-/- mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b-CD103- double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses.
Asunto(s)
Ligando de CD40/genética , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/terapia , Receptores Quiméricos de Antígenos/genética , Inmunidad Adaptativa , Animales , Presentación de Antígeno , Antígenos CD/genética , Antígenos CD/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Antígeno CD11b/deficiencia , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Ligando de CD40/inmunología , Linfocitos T CD8-positivos/citología , Células Dendríticas/citología , Femenino , Expresión Génica , Inmunidad Innata , Inmunofenotipificación , Cadenas alfa de Integrinas/deficiencia , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/inmunología , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Trasplante de Neoplasias , Receptores Quiméricos de Antígenos/inmunología , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Proteínas Represoras/inmunologíaRESUMEN
CAR T-cell therapy for multiple myeloma (MM) targeting B-cell maturation antigen (TNFRSF17; BCMA) induces high overall response rates; however, relapse occurs commonly. Implicated in relapse is a reservoir of MM if cells lacking sufficient BCMA surface expression (antigen escape). We demonstrate that simultaneous targeting of an additional antigen-here, G protein-coupled receptor class-C group-5 member-D (GPRC5D)-can prevent BCMA escape-mediated relapse in a model of MM. To identify an optimal approach, we compare subtherapeutic doses of different forms of dual-targeted cellular therapy. These include (1) parallel-produced and pooled mono-targeted CAR T-cells, (2) bicistronic constructs expressing distinct CARs from a single vector, and (3) a dual-scFv "single-stalk" CAR design. When targeting BCMA-negative disease, bicistronic and pooled approaches had the highest efficacy, whereas for dual-antigen-expressing disease, the bicistronic approach was more efficacious than the pooled approach. Mechanistically, expressing two CARs on a single cell enhanced the strength of CAR T-cell/target cell interactions.
Asunto(s)
Inmunoterapia Adoptiva , Mieloma Múltiple , Antígeno de Maduración de Linfocitos B/genética , Humanos , Mieloma Múltiple/genética , Recurrencia Local de Neoplasia , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Although clinical responses with CD19-targeting chimeric antigen receptor (CAR) T-cell treatment have been observed in patients with certain hematologic malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Because T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3ζ signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19-targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR-mediated CD28 costimulation. Costimulation-driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3ζ), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation.See related article by Drakes et al., p. 743.
Asunto(s)
Antígenos CD28/inmunología , Inmunoterapia Adoptiva/métodos , Interleucina-12/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Timoma/inmunología , Neoplasias del Timo/inmunología , Animales , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Apoptosis , Antígenos CD28/metabolismo , Proliferación Celular , Citocinas , Humanos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Timoma/metabolismo , Timoma/patología , Neoplasias del Timo/metabolismo , Neoplasias del Timo/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
T-cell receptor (TCR)-modified T-cell gene therapy can target a variety of extracellular and intracellular tumor-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo We postulated that expression of proinflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior proinflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a proinflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18-secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1-reactive TCR-modified human T cells as well as overall survival of tumor-bearing mice. These results demonstrate a rationale for optimizing the efficacy of TCR-modified T-cell cancer therapy through expression of IL18.See related commentary by Wijewarnasuriya et al., p. 732.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia Adoptiva/normas , Interleucina-12/metabolismo , Interleucina-18/metabolismo , Activación de Linfocitos/inmunología , Melanoma/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/inmunología , Apoptosis , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Humanos , Inmunoterapia Adoptiva/métodos , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/patología , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones Noqueados , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chimeric antigen receptor (CAR) T cells provide great efficacy in B cell malignancies. However, improved CAR T cell therapies are still needed. Here, we engineered tumor-targeted CAR T cells to constitutively express the immune-stimulatory molecule CD40 ligand (CD40L) and explored efficacy in different mouse leukemia/lymphoma models. We observed that CD40L+ CAR T cells circumvent tumor immune escape via antigen loss through CD40/CD40L-mediated cytotoxicity and induction of a sustained, endogenous immune response. After adoptive cell transfer, the CD40L+ CAR T cells displayed superior antitumor efficacy, licensed antigen-presenting cells, enhanced recruitment of immune effectors, and mobilized endogenous tumor-recognizing T cells. These effects were absent in Cd40-/- mice and provide a rationale for the use of CD40L+ CAR T cells in cancer treatment.
Asunto(s)
Ligando de CD40/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Ligando de CD40/genética , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Receptores Quiméricos de Antígenos/genética , Escape del Tumor , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.