RESUMO
Interleukin-15 (IL15) promotes the survival of T lymphocytes and enhances the antitumor properties of CAR T cells in preclinical models of solid neoplasms in which CAR T cells have limited efficacy1-4. Glypican-3 (GPC3) is expressed in a group of solid cancers5-10, and here we report the first evaluation in humans of the effects of IL15 co-expression on GPC3-CAR T cells. Cohort 1 patients (NCT02905188/NCT02932956) received GPC3-CAR T cells, which were safe but produced no objective antitumor responses and reached peak expansion at two weeks. Cohort 2 patients (NCT05103631/NCT04377932) received GPC3-CAR T cells that co-expressed IL15 (15.CAR), which mediated significantly increased cell expansion and induced a disease control rate of 66% and antitumor response rate of 33%. Infusion of 15.CAR T cells was associated with increased incidence of cytokine release syndrome, which was rapidly ameliorated by activation of the inducible caspase 9 safety switch. Compared to non-responders, tumor-infiltrating 15.CAR T cells from responders showed repression of SWI/SNF epigenetic regulators and upregulation of FOS and JUN family members as well as genes related to type I interferon signaling. Collectively, these results demonstrate that IL15 increases the expansion, intratumoral survival, and antitumor activity of GPC3-CAR T cells in patients.
RESUMO
Human natural killer T cells (NKTs) are innate-like T lymphocytes increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintains a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observe that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates a de novo programming of memory NKTs in CD62L-negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy, and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 show enhanced antitumor activity in leukemia and neuroblastoma tumor models, persist long-term in vivo and conserve the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics.
Assuntos
Células T Matadoras Naturais , Receptores de Antígenos Quiméricos , Humanos , Interleucina-12/genética , Citotoxicidade Imunológica , Ativação LinfocitáriaRESUMO
Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Domínios Proteicos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T , Neoplasias/metabolismo , Imunoglobulina G/metabolismo , Imunoterapia Adotiva/métodos , Antígenos CD19RESUMO
Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .
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Células T Matadoras Naturais , Neuroblastoma , Receptores de Antígenos Quiméricos , Criança , Animais , Camundongos , Humanos , Citotoxicidade Imunológica , Receptores de Antígenos Quiméricos/genética , Neuroblastoma/terapia , Imunoterapia Adotiva/métodosRESUMO
BACKGROUND: Tumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the mechanism by which tumor-supportive inflammation is initiated and propagated remains unknown. Here, we describe a novel protumorigenic circuit between NB cells and monocytes that is triggered and sustained by tumor necrosis factor alpha (TNF-α). METHODS: We used NB knockouts (KOs) of TNF-α and TNFRSF1A mRNA (TNFR1)/TNFRSF1B mRNA (TNFR2) and TNF-α protease inbitor (TAPI), a drug that modulates TNF-α isoform expression, to assess the role of each component in monocyte-associated protumorigenic inflammation. Additionally, we employed NB-monocyte cocultures and treated these with clinical-grade etanercept, an Fc-TNFR2 fusion protein, to neutralize signaling by both membrane-bound (m) and soluble (s)TNF-α isoforms. Further, we treated NOD/SCID/IL2Rγ(null) mice carrying subcutaneous NB/human monocyte xenografts with etanercept and evaluated the impact on tumor growth and angiogenesis. Gene set enrichment analysis (GSEA) was used to determine whether TNF-α signaling correlates with clinical outcomes in patients with NB. RESULTS: We found that NB expression of TNFR2 and monocyte membrane-bound tumor necrosis factor alpha is required for monocyte activation and interleukin (IL)-6 production, while NB TNFR1 and monocyte soluble TNF-α are required for NB nuclear factor kappa B subunit 1 (NF-κB) activation. Treatment of NB-monocyte cocultures with clinical-grade etanercept completely abrogated release of IL-6, granulocyte colony-stimulating factor (G-CSF), IL-1α, and IL-1ß and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNF-α signaling in patients with NB that relapsed. CONCLUSIONS: We have described a novel mechanism of tumor-promoting inflammation in NB that is strongly associated with patient outcome and could be targeted with therapy.
Assuntos
Neuroblastoma , Receptores Tipo II do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa , Animais , Humanos , Camundongos , Carcinogênese , Etanercepte , Camundongos Endogâmicos NOD , Camundongos SCID , Monócitos , Neuroblastoma/tratamento farmacológico , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/genéticaRESUMO
Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L- cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/ß-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/ß-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/ß-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory-like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell-based immunotherapy. See related Spotlight by Van Kaer, p. 144.
Assuntos
Células T Matadoras Naturais , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Células T Matadoras Naturais/imunologia , beta Catenina , Fator 1 de Ligação ao Facilitador Linfoide/genética , Ativação Linfocitária/imunologiaRESUMO
T cells expressing chimeric antigen receptors (CARs) have achieved major clinical success in patients with hematologic malignancies. However, these treatments remain largely ineffective for solid cancers and require significant time and resources to be manufactured in an autologous setting. Developing alternative immune effector cells as cancer immunotherapy agents that can be employed in allogeneic settings is crucial for the advancement of cell therapy. Unlike T cells, Vα24-invariant natural killer T cells (NKTs) are not alloreactive and can therefore be generated from allogeneic donors for rapid infusion into numerous patients without the risk of graft-versus-host disease. Additionally, NKT cells demonstrate inherent advantages over T-cell products, including the ability to traffic to tumor tissues, target tumor-associated macrophages, transactivate NK cells, and cross-prime tumor-specific CD8 T cells. Both unmodified NKTs, which specifically recognize CD1d-bound glycolipid antigens expressed by certain types of tumors, and CAR-redirected NKTs are being developed as the next generation of allogeneic cell therapy products. In this review, we describe studies on the biology of NKTs and other types of innate-like T cells and summarize the clinical experiences of unmodified and CAR-redirected NKTs, including recent interim reports on allogeneic NKTs.
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Transplante de Células-Tronco Hematopoéticas , Células T Matadoras Naturais , Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Células Alógenas , Neoplasias/terapia , Células Matadoras Naturais , Imunoterapia AdotivaRESUMO
Subsequent malignancies are well-documented complications in long-term follow-up of cancer patients. Recently, genetically modified immune effector (IE) cells have shown benefit in hematologic malignancies and are being evaluated in clinical trials for solid tumors. Although the short-term complications of IE cells are well described, there is limited literature summarizing long-term follow-up, including subsequent malignancies. We retrospectively reviewed data from 340 patients treated across 27 investigator-initiated pediatric and adult clinical trials at our center. All patients received IE cells genetically modified with γ-retroviral vectors to treat relapsed and/or refractory hematologic or solid malignancies. In a cumulative 1027 years of long-term follow-up, 13 patients (3.8%) developed another cancer with a total of 16 events (4 hematologic malignancies and 12 solid tumors). The 5-year cumulative incidence of a first subsequent malignancy in the recipients of genetically modified IE cells was 3.6% (95% confidence interval, 1.8% to 6.4%). For 11 of the 16 subsequent tumors, biopsies were available, and no sample was transgene positive by polymerase chain reaction. Replication-competent retrovirus testing of peripheral blood mononuclear cells was negative in the 13 patients with subsequent malignancies tested. Rates of subsequent malignancy were low and comparable to standard chemotherapy. These results suggest that the administration of IE cells genetically modified with γ retroviral vectors does not increase the risk for subsequent malignancy.
Assuntos
Neoplasias Hematológicas , Neoplasias , Adulto , Criança , Seguimentos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Leucócitos Mononucleares , Neoplasias/genética , Neoplasias/terapia , Estudos RetrospectivosRESUMO
Objective: Tumor-associated macrophages (TAMs) within the tumor immune microenvironment (TiME) of solid tumors play an important role in treatment resistance and disease recurrence. The purpose of this study was to investigate if nanoradiomics (radiomic analysis of nanoparticle contrast-enhanced images) can differentiate tumors based on TAM burden. Materials and Methods: In vivo studies were performed in transgenic mouse models of neuroblastoma with low (N = 11) and high (N = 10) tumor-associated macrophage (TAM) burden. Animals underwent delayed nanoparticle contrast-enhanced CT (n-CECT) imaging at 4 days after intravenous administration of liposomal-iodine agent (1.1 g/kg). CT imaging-derived conventional tumor metrics (tumor volume and CT attenuation) were computed for segmented tumor CT datasets. Nanoradiomic analysis was performed using a PyRadiomics workflow implemented in the quantitative image feature pipeline (QIFP) server containing 900 radiomic features (RFs). RF selection was performed under supervised machine learning using a nonparametric neighborhood component method. A 5-fold validation was performed using a set of linear and nonlinear classifiers for group separation. Statistical analysis was performed using the Kruskal-Wallis test. Results: N-CECT imaging demonstrated heterogeneous patterns of signal enhancement in low and high TAM tumors. CT imaging-derived conventional tumor metrics showed no significant differences (p > 0.05) in tumor volume between low and high TAM tumors. Tumor CT attenuation was not significantly different (p > 0.05) between low and high TAM tumors. Machine learning-augmented nanoradiomic analysis revealed two RFs that differentiated (p < 0.002) low TAM and high TAM tumors. The RFs were used to build a linear classifier that demonstrated very high accuracy and further confirmed by 5-fold cross-validation. Conclusions: Imaging-derived conventional tumor metrics were unable to differentiate tumors with varying TAM burden; however, nanoradiomic analysis revealed texture differences and enabled differentiation of low and high TAM tumors.
Assuntos
Meios de Contraste/farmacologia , Nanopartículas/química , Neuroblastoma/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Animais , Meios de Contraste/química , Humanos , Radioisótopos do Iodo/química , Radioisótopos do Iodo/farmacologia , Aprendizado de Máquina , Camundongos , Camundongos Transgênicos , Neuroblastoma/patologia , Radiometria , Carga Tumoral/efeitos da radiação , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos da radiação , Macrófagos Associados a TumorRESUMO
Vα24-invariant natural killer T (NKT) cells have shown potent anti-tumor properties in murine tumor models and have been linked to favorable outcomes in patients with cancer. However, low numbers of these cells in humans have hindered their clinical applications. Here we report interim results from all three patients enrolled on dose level 1 in a phase 1 dose-escalation trial of autologous NKT cells engineered to co-express a GD2-specific chimeric antigen receptor (CAR) with interleukin-15 in children with relapsed or resistant neuroblastoma (NCT03294954). Primary and secondary objectives were to assess safety and anti-tumor responses, respectively, with immune response evaluation as an additional objective. We ex vivo expanded highly pure NKT cells (mean ± s.d., 94.7 ± 3.8%) and treated patients with 3 × 106 CAR-NKT cells per square meter of body surface area after lymphodepleting conditioning with cyclophosphamide/fludarabine (Cy/Flu). Cy/Flu conditioning was the probable cause for grade 3-4 hematologic adverse events, as they occurred before CAR-NKT cell infusion, and no dose-limiting toxicities were observed. CAR-NKT cells expanded in vivo, localized to tumors and, in one patient, induced an objective response with regression of bone metastatic lesions. These initial results suggest that CAR-NKT cells can be expanded to clinical scale and safely applied to treat patients with cancer.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Células T Matadoras Naturais/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Receptores de Antígenos Quiméricos/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Criança , Ciclofosfamida/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Imunidade/efeitos dos fármacos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Masculino , Células T Matadoras Naturais/imunologia , Neuroblastoma/genética , Neuroblastoma/imunologia , Neuroblastoma/patologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Vidarabina/administração & dosagem , Vidarabina/análogos & derivadosRESUMO
Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death in the world, and curative systemic therapies are lacking. Chimeric antigen receptor (CAR)-expressing T cells induce robust antitumor responses in patients with hematologic malignancies but have limited efficacy in patients with solid tumors, including HCC. IL15 and IL21 promote T-cell expansion, survival, and function and can improve the antitumor properties of T cells. We explored whether transgenic expression of IL15 and/or IL21 enhanced glypican-3-CAR (GPC3-CAR) T cells' antitumor properties against HCC. We previously optimized the costimulation in GPC3-CARs and selected a second-generation GPC3-CAR incorporating a 4-1BB costimulatory endodomain (GBBz) for development. Here, we generated constructs encoding IL15, IL21, or both with GBBz (15.GBBz, 21.GBBz, and 21.15.GBBz, respectively) and examined the ability of transduced T cells to kill, produce effector cytokines, and expand in an antigen-dependent manner. We performed gene-expression and phenotypic analyses of GPC3-CAR T cells and CRISPR-Cas9 knockout of the TCF7 gene. Finally, we measured GPC3-CAR T-cell antitumor activity in murine xenograft models of GPC3+ tumors. The increased proliferation of 21.15.GBBz T cells was at least in part dependent on the upregulation and maintenance of TCF-1 (encoded by TCF7) and associated with a higher percentage of stem cell memory and central memory populations after manufacturing. T cells expressing 21.15.GBBz had superior in vitro and in vivo expansion and persistence, and the most robust antitumor activity in vivo These results provided preclinical evidence to support the clinical evaluation of 21.15.GPC3-CAR T cells in patients with HCC.
Assuntos
Carcinoma Hepatocelular/terapia , Glipicanas/imunologia , Imunoterapia Adotiva/métodos , Interleucina-15/imunologia , Interleucinas/imunologia , Neoplasias Hepáticas/terapia , Animais , Apoptose/imunologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/fisiologia , Feminino , Glipicanas/genética , Humanos , Interleucina-15/biossíntese , Interleucina-15/genética , Interleucinas/biossíntese , Interleucinas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T-cell receptor (TCR) gene transfer redirects T cells to target intracellular antigens. However, the potential autoreactivity generated by TCR mispairing and occurrence of graft-versus-host disease in the allogenic setting due to the retention of native TCRs remain major concerns. Natural killer T cells (NKT) have shown promise as a platform for adoptive T-cell therapy in cancer patients. Here, we showed their utility for TCR gene transfer. We successfully engineered and expanded NKTs expressing a functional TCR (TCR NKTs), showing HLA-restricted antitumor activity in xenogeneic mouse models in the absence of graft-versus-mouse reactions. We found that TCR NKTs downregulated the invariant TCR (iTCR), leading to iTCR+TCR+ and iTCR-TCR+ populations. In-depth analyses of these subsets revealed that in iTCR-TCR+ NKTs, the iTCR, although expressed at the mRNA and protein levels, was retained in the cytoplasm. This effect resulted from a competition for binding to CD3 molecules for cell-surface expression by the transgenic TCR. Overall, our results highlight the feasibility and advantages of using NKTs for TCR expression for adoptive cell immunotherapies. NKT-low intrinsic alloreactivity that associated with the observed iTCR displacement by the engineered TCR represents ideal characteristics for "off-the-shelf" products without further TCR gene editing.
Assuntos
Complexo CD3/imunologia , Citotoxicidade Imunológica/imunologia , Imunoterapia Adotiva/métodos , Melanoma/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/classificação , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Camundongos Endogâmicos NOD , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Vα24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs. EXPERIMENTAL DESIGN: Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma. RESULTS: Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activation-induced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2.CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround in vitro tumor cell killing. Following transfer into mice bearing neuroblastoma xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared with GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathologic analysis. CONCLUSIONS: Our results informed selection of the CD28-based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).
Assuntos
Citotoxicidade Imunológica/imunologia , Gangliosídeos/imunologia , Imunoterapia Adotiva/métodos , Interleucina-15/imunologia , Células T Matadoras Naturais/transplante , Neuroblastoma/terapia , Receptores de Antígenos Quiméricos/imunologia , Animais , Apoptose , Proliferação de Células , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células T Matadoras Naturais/imunologia , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Solid tumors are refractory to cellular immunotherapies in part because they contain suppressive immune effectors such as myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic lymphocytes. Strategies to reverse the suppressive tumor microenvironment (TME) should also attract and activate immune effectors with antitumor activity. To address this need, we developed gene-modified natural killer (NK) cells bearing a chimeric receptor in which the activating receptor NKG2D is fused to the cytotoxic ζ-chain of the T-cell receptor (NKG2D.ζ). NKG2D.ζ-NK cells target MDSCs, which overexpress NKG2D ligands within the TME. We examined the ability of NKG2D.ζ-NK cells to eliminate MDSCs in a xenograft TME model and improve the antitumor function of tumor-directed chimeric antigen receptor (CAR)-modified T cells. We show that NKG2D.ζ-NK cells are cytotoxic against MDSCs, but spare NKG2D ligand-expressing normal tissues. NKG2D.ζ-NK cells, but not unmodified NK cells, secrete proinflammatory cytokines and chemokines in response to MDSCs at the tumor site and improve infiltration and antitumor activity of subsequently infused CAR-T cells, even in tumors for which an immunosuppressive TME is an impediment to treatment. Unlike endogenous NKG2D, NKG2D.ζ is not susceptible to TME-mediated downmodulation and thus maintains its function even within suppressive microenvironments. As clinical confirmation, NKG2D.ζ-NK cells generated from patients with neuroblastoma killed autologous intratumoral MDSCs capable of suppressing CAR-T function. A combination therapy for solid tumors that includes both NKG2D.ζ-NK cells and CAR-T cells may improve responses over therapies based on CAR-T cells alone.
Assuntos
Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Supressoras Mieloides/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Humanos , Imunoterapia Adotiva , Células K562 , Ligantes , Camundongos , Células Supressoras Mieloides/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neuroblastoma/imunologia , Neuroblastoma/patologia , Neuroblastoma/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/administração & dosagem , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Peptídeos/administração & dosagem , Adjuvantes Imunológicos , Animais , Antígenos CD1d/química , Vacinas Anticâncer/imunologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Epitopos/química , Glicolipídeos/química , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/tratamento farmacológico , Peptídeos/química , Peptídeos/imunologiaRESUMO
T cells expressing CD19-specific chimeric Ag receptors (CARs) produce high remission rates in B cell lymphoma, but frequent disease recurrence and challenges in generating sufficient numbers of autologous CAR T cells necessitate the development of alternative therapeutic effectors. Vα24-invariant NKTs have intrinsic antitumor properties and are not alloreactive, allowing for off-the-shelf use of CAR-NKTs from healthy donors. We recently reported that CD62L+ NKTs persist longer and have more potent antilymphoma activity than CD62L- cells. However, the conditions governing preservation of CD62L+ cells during NKT cell expansion remain largely unknown. In this study, we demonstrate that IL-21 preserves this crucial central memory-like NKT subset and enhances its antitumor effector functionality. We found that following antigenic stimulation with α-galactosylceramide, CD62L+ NKTs both expressed IL-21R and secreted IL-21, each at significantly higher levels than CD62L- cells. Although IL-21 alone failed to expand stimulated NKTs, combined IL-2/IL-21 treatment produced more NKTs and increased the frequency of CD62L+ cells versus IL-2 alone. Gene expression analysis comparing CD62L+ and CD62L- cells treated with IL-2 alone or IL-2/IL-21 revealed that the latter condition downregulated the proapoptotic protein BIM selectively in CD62L+ NKTs, protecting them from activation-induced cell death. Moreover, IL-2/IL-21-expanded NKTs upregulated granzyme B expression and produced more TH1 cytokines, leading to enhanced in vitro cytotoxicity of nontransduced and anti-CD19-CAR-transduced NKTs against CD1d+ and CD19+ lymphoma cells, respectively. Further, IL-2/IL-21-expanded CAR-NKTs dramatically increased the survival of lymphoma-bearing NSG mice compared with IL-2-expanded CAR-NKTs. These findings have immediate translational implications for the development of NKT cell-based immunotherapies targeting lymphoma and other malignancies.
Assuntos
Imunoterapia Adotiva/métodos , Interleucinas/metabolismo , Linfoma de Células B/terapia , Células T Matadoras Naturais/imunologia , Células Th1/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica , Galactosilceramidas/imunologia , Granzimas/metabolismo , Humanos , Interleucina-2/metabolismo , Selectina L/metabolismo , Ativação Linfocitária , Linfoma de Células B/imunologia , Camundongos , Células T Matadoras Naturais/transplante , Transplante de Neoplasias , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer.Significance: The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. Cancer Discov; 7(11); 1238-47. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.
Assuntos
Glioblastoma/terapia , Imunoterapia Adotiva , Interleucina-7/imunologia , Neuroblastoma/terapia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Glioblastoma/genética , Glioblastoma/imunologia , Humanos , Interleucina-7/genética , Camundongos , Neuroblastoma/genética , Neuroblastoma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/uso terapêutico , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia , Receptores de Citocinas/uso terapêutico , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3ζ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon γ/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.