RESUMEN
Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.
Asunto(s)
Sangre Fetal/citología , Inmunoterapia Adoptiva , Interleucina-15/genética , Células Asesinas Naturales/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Supresoras de la Señalización de Citocinas/antagonistas & inhibidores , Aerobiosis , Animales , Antígenos CD19/inmunología , Linfoma de Burkitt/patología , Linfoma de Burkitt/terapia , Sistemas CRISPR-Cas , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/trasplante , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores Quiméricos de Antígenos , Transducción de Señal/fisiología , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chimeric antigen receptor (CAR) T cell show promise in cancer treatments, but their mechanism of action is not well understood. Decoding the mechanisms used by individual T cells can help improve the efficacy of T cells while also identifying mechanisms of T cell failure leading to tumor escape. Here, we used a suite of assays including dynamic single-cell imaging of cell-cell interactions, dynamic imaging of fluorescent reporters to directly track cytotoxin activity in tumor cells, and scRNA-seq on patient infusion products to investigate the cytotoxic mechanisms used by individual CAR T cells in killing tumor cells. We show that surprisingly, overexpression of the Granzyme B (GZMB) inhibitor, protease inhibitor-9 (PI9), does not alter the cytotoxicity mediated by CD19-specific CAR T cells against either the leukemic cell line, NALM6; or the ovarian cancer cell line, SkOV3-CD19. We designed and validated reporters to directly assay T cell delivered GZMB activity in tumor cells and confirmed that while PI9 overexpression inhibits GZMB activity at the molecular level, this is not sufficient to impact the kinetics or magnitude of killing mediated by the CAR T cells. Altering cytotoxicity mediated by CAR T cells required combined inhibition of multiple pathways that are tumor cell specific: (a) B-cell lines like NALM6, Raji and Daudi were sensitive to combined GZMB and granzyme A (GZMA) inhibition; whereas (b) solid tumor targets like SkOV3-CD19 and A375-CD19 (melanoma) were sensitive to combined GZMB and Fas ligand inhibition. We realized the translational relevance of these findings by examining the scRNA-seq profiles of Tisa-cel and Axi-cel infusion products and show a significant correlation between GZMB and GZMA expression at the single-cell level in a T cell subset-dependent manner. Our findings highlight the importance of the redundancy in killing mechanisms of CAR T cells and how this redundancy is important for efficacious T cells.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Granzimas/genética , Linfocitos T , Inmunoterapia Adoptiva/métodosRESUMEN
Immunization programs against SARS-CoV-2 with commercial intramuscular vaccines prevent disease but are less efficient in preventing infections. Mucosal vaccines can provide improved protection against transmission, ideally for different variants of concern (VOCs) and related sarbecoviruses. Here, we report a multi-antigen, intranasal vaccine, NanoSTING-SN (NanoSTING-Spike-Nucleocapsid), eliminates virus replication in both the lungs and the nostrils upon challenge with the pathogenic SARS-CoV-2 Delta VOC. We further demonstrate that NanoSTING-SN prevents transmission of the SARS-CoV-2 Omicron VOC (BA.5) to vaccine-naïve hamsters. To evaluate protection against other sarbecoviruses, we immunized mice with NanoSTING-SN. We showed that immunization affords protection against SARS-CoV, leading to protection from weight loss and 100% survival in mice. In non-human primates, animals immunized with NanoSTING-SN show durable serum IgG responses (6 months) and nasal wash IgA responses cross-reactive to SARS-CoV-2 (XBB1.5), SARS-CoV and MERS-CoV antigens. These observations have two implications: (1) mucosal multi-antigen vaccines present a pathway to reducing transmission of respiratory viruses, and (2) eliciting immunity against multiple antigens can be advantageous in engineering pan-sarbecovirus vaccines.
Asunto(s)
Administración Intranasal , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Animales , SARS-CoV-2/inmunología , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/transmisión , COVID-19/virología , Ratones , Cricetinae , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Femenino , Ratones Endogámicos BALB C , Humanos , Mesocricetus , Antígenos Virales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangreRESUMEN
Chimeric antigen receptor (CAR) T cells used for the treatment of B cell malignancies can identify T cell subsets with superior clinical activity. Here, using infusion products of individuals with large B cell lymphoma, we integrated functional profiling using timelapse imaging microscopy in nanowell grids with subcellular profiling and single-cell RNA sequencing to identify a signature of multifunctional CD8+ T cells (CD8-fit T cells). CD8-fit T cells are capable of migration and serial killing and harbor balanced mitochondrial and lysosomal volumes. Using independent datasets, we validate that CD8-fit T cells (1) are present premanufacture and are associated with clinical responses in individuals treated with axicabtagene ciloleucel, (2) longitudinally persist in individuals after treatment with CAR T cells and (3) are tumor migrating cytolytic cells capable of intratumoral expansion in solid tumors. Our results demonstrate the power of multimodal integration of single-cell functional assessments for the discovery and application of CD8-fit T cells as a T cell subset with optimal fitness in cell therapy.
Asunto(s)
Linfocitos T CD8-positivos , Inmunoterapia Adoptiva , Linfoma de Células B Grandes Difuso , Receptores Quiméricos de Antígenos , Análisis de la Célula Individual , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/terapia , Análisis de la Célula Individual/métodos , Inmunoterapia Adoptiva/métodos , Linfocitos T CD8-positivos/inmunología , Receptores Quiméricos de Antígenos/inmunología , Subgrupos de Linfocitos T/inmunología , Productos BiológicosRESUMEN
Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3Z creates a platform that recruits critical kinases, such as LCK and ZAP70, initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy.
RESUMEN
Extracellular vesicles (EVs) regulate the tumor microenvironment by facilitating transport of biomolecules. Despite extensive investigation, heterogeneity in EV secretion among cancer cells and the mechanisms that support EV secretion are not well characterized. We developed an integrated method to identify individual cells with differences in EV secretion and performed linked single-cell RNA-sequencing on cloned single cells from the metastatic breast cancer cells. Differential gene expression analyses identified a four-gene signature of breast cancer EV secretion: HSP90AA1, HSPH1, EIF5, and DIAPH3. We functionally validated this gene signature by testing it across cell lines with different metastatic potential in vitro. Analysis of the TCGA and METABRIC datasets showed that this signature is associated with poor survival, invasive breast cancer types, and poor CD8+ T cell infiltration in human tumors. We anticipate that our method for directly identifying the molecular determinants of EV secretion will have broad applications across cell types and diseases.
RESUMEN
The in vivo persistence of adoptively transferred T cells is predictive of antitumor response. Identifying functional properties of infused T cells that lead to in vivo persistence and tumor eradication has remained elusive. We profiled CD19-specific chimeric antigen receptor (CAR) T cells as the infusion products used to treat large B cell lymphomas using high-throughput single-cell technologies based on time-lapse imaging microscopy in nanowell grids (TIMING), which integrates killing, cytokine secretion, and transcriptional profiling. Our results show that the directional migration of CD19-specific CAR T cells is correlated with multifunctionality. We showed that CD2 on T cells is associated with directional migration and that the interaction between CD2 on T cells and CD58 on lymphoma cells accelerates killing and serial killing. Consistent with this, we observed that elevated CD58 expression on pretreatment tumor samples in patients with relapsed or refractory large B cell lymphomas treated with CD19-specific CAR T cell therapy was associated with complete clinical response and survival. These results highlight the importance of studying dynamic T cell-tumor cell interactions in identifying optimal antitumor responses.
Asunto(s)
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Linfoma de Células B Grandes Difuso , Linfocitos T , Antígenos CD19 , Humanos , Inmunoterapia Adoptiva/métodos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/terapia , Receptores de Antígenos de Linfocitos T , Análisis de la Célula IndividualRESUMEN
Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.
Asunto(s)
Receptores Quiméricos de Antígenos , Antígenos de Neoplasias , Línea Celular Tumoral , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales , Receptores Quiméricos de Antígenos/metabolismo , Trogocitosis , Escape del TumorRESUMEN
Despite remarkable progress in the development and authorization of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is a need to validate vaccine platforms for broader application. The current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity in the nasal compartment, which is the first barrier that SARS-CoV-2 virus breaches before dissemination to the lung. We report the development of an intranasal subunit vaccine that uses lyophilized spike protein and liposomal STING agonist as an adjuvant. This vaccine induces systemic neutralizing antibodies, IgA in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA sequencing confirmed the coordinated activation of T/B-cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues, confirming its role as an inductive site to enable durable immunity. The ability to elicit immunity in the respiratory tract can prevent the establishment of infection in individuals and prevent disease transmission.
RESUMEN
A safe and durable vaccine is urgently needed to tackle the COVID19 pandemic that has infected >15 million people and caused >620,000 deaths worldwide. As with other respiratory pathogens, the nasal compartment is the first barrier that needs to be breached by the SARS-CoV-2 virus before dissemination to the lung. Despite progress at remarkable speed, current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. We report the development of an intranasal subunit vaccine that contains the trimeric or monomeric spike protein and liposomal STING agonist as adjuvant. This vaccine induces systemic neutralizing antibodies, mucosal IgA responses in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA-sequencing confirmed the concomitant activation of T and B cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues (NALT), confirming its role as an inductive site that can lead to long-lasting immunity. The ability to elicit immunity in the respiratory tract has can prevent the initial establishment of infection in individuals and prevent disease transmission across humans.
RESUMEN
Time lapse microscopy is essential for quantifying the dynamics of cells, subcellular organelles and biomolecules. Biologists use different fluorescent tags to label and track the subcellular structures and biomolecules within cells. However, not all of them are compatible with time lapse imaging, and the labeling itself can perturb the cells in undesirable ways. We hypothesized that phase image has the requisite information to identify and track nuclei within cells. By utilizing both traditional blob detection to generate binary mask labels from the stained channel images and the deep learning Mask RCNN model to train a detection and segmentation model, we managed to segment nuclei based only on phase images. The detection average precision is 0.82 when the IoU threshold is to be set 0.5. And the mean IoU for masks generated from phase images and ground truth masks from experts is 0.735. Without any ground truth mask labels during the training time, this is good enough to prove our hypothesis. This result enables the ability to detect nuclei without the need for exogenous labeling.