An innovative single-cell approach for phenotyping and functional genotyping of CAR NK cells.

BACKGROUND: To accelerate the translation of novel immunotherapeutic treatment approaches, the development of analytic methods to assess their efficacy at early in vitro stages is necessary. Using a droplet-based microfluidic platform, we have established a method for multiparameter quantifiable phenotypic and genomic observations of immunotherapies. Chimeric antigen receptor (CAR) natural killer (NK) cells are of increased interest in the current immunotherapy landscape and thus provide an optimal model for evaluating our novel methodology. METHODS: For this approach, NK cells transduced with a CD19 CAR were compared with non-transduced NK cells in their ability to kill a lymphoma cell line. Using our microfluidic platform, we were able to quantify the increase in cytotoxicity and synaptic contact formation of CAR NK cells over non-transduced NK cells. We then optimized our droplet sorter and successfully used it to separate NK cells based on target cell killing to perform transcriptomic analyses. RESULTS: Our data revealed expected improvement in cytotoxicity with the CD19 CAR but more importantly, provided unique insights into the factors involved in the cytotoxic mechanisms of CAR NK cells. This demonstrates a novel, improved system for accelerating the pre-clinical screening of future immunotherapy treatments. CONCLUSIONS: This study provides a new potential approach for enhanced early screening of immunotherapies to improve their development, with a highly relevant cell model to demonstrate. Additionally, our validation studies provided some potential insights into transcriptomic determinants influencing CAR NK cytotoxicity.

CRK and NCK adaptors may functionally overlap in zebrafish neurodevelopment, as indicated by common binding partners and overlapping expression patterns.

CRK adaptor proteins are important for signal transduction mechanisms driving cell proliferation and positioning during vertebrate central nervous system development. Zebrafish lacking both CRK family members exhibit small, disorganized retinas with 50% penetrance. The goal of this study was to determine whether another adaptor protein might functionally compensate for the loss of CRK adaptors. Expression patterns in developing zebrafish, and bioinformatic analyses of the motifs recognized by their SH2 and SH3 domains, suggest NCK adaptors are well-positioned to compensate for loss of CRK adaptors. In support of this hypothesis, proteomic analyses found CRK and NCK adaptors share overlapping interacting partners including known regulators of cell adhesion and migration, suggesting their functional intersection in neurodevelopment.

Non-pathogenic E. coli displaying decoy-resistant IL18 mutein boosts anti-tumor and CAR NK cell responses.

The tumor microenvironment can inhibit the efficacy of cancer therapies through mechanisms such as poor trafficking and exhaustion of immune cells. Here, to address this challenge, we exploited the safety, tumor tropism and ease of genetic manipulation of non-pathogenic Escherichia coli (E. coli) to deliver key immune-activating cytokines to tumors via surface display on the outer membrane of E. coli K-12 DH5α. Non-pathogenic E. coli expressing murine decoy-resistant IL18 mutein (DR18) induced robust CD8+ T and natural killer (NK) cell-dependent immune responses and suppressed tumor progression in immune-competent colorectal carcinoma and melanoma mouse models. E. coli K-12 DH5α engineered to display human DR18 potently activated mesothelin-targeting chimeric antigen receptor (CAR) NK cells and enhance their trafficking into tumors, which extended survival in an NK cell treatment-resistant mesothelioma xenograft model by enhancing TNF signaling and upregulating NK activation markers. Our live bacteria-based immunotherapeutic system safely and effectively induces potent anti-tumor responses in treatment-resistant solid tumors, motivating further evaluation of this approach in the clinic.

Nonpathogenic E. coli engineered to surface display cytokines as a new platform for immunotherapy.

Given the safety, tumor tropism, and ease of genetic manipulation in non-pathogenic Escherichia coli (E. coli), we designed a novel approach to deliver biologics to overcome poor trafficking and exhaustion of immune cells in the tumor microenvironment, via the surface display of key immune-activating cytokines on the outer membrane of E. coli K-12 DH5α. Bacteria expressing murine decoy-resistant IL18 mutein (DR18) induced robust CD8+ T and NK cell-dependent immune responses leading to dramatic tumor control, extending survival, and curing a significant proportion of immune-competent mice with colorectal carcinoma and melanoma. The engineered bacteria demonstrated tumor tropism, while the abscopal and recall responses suggested epitope spreading and induction of immunologic memory. E. coli K-12 DH5α engineered to display human DR18 potently activated mesothelin-targeting CAR NK cells and safely enhanced their trafficking into the tumors, leading to improved control and survival in xenograft mice bearing mesothelioma tumor cells, otherwise resistant to NK cells. Gene expression analysis of the bacteria-primed CAR NK cells showed enhanced TNFα signaling via NFkB and upregulation of multiple activation markers. Our novel live bacteria-based immunotherapeutic platform safely and effectively induces potent anti-tumor responses in otherwise hard-to-treat solid tumors, motivating further evaluation of this approach in the clinic.

CAR memory-like NK cells targeting the membrane proximal domain of mesothelin demonstrate promising activity in ovarian cancer.

Epithelial ovarian cancer (EOC) remains one of the most lethal gynecological cancers. Cytokine-induced memory-like (CIML) natural killer (NK) cells have shown promising results in preclinical and early-phase clinical trials. In the current study, CIML NK cells demonstrated superior antitumor responses against a panel of EOC cell lines, increased expression of activation receptors, and up-regulation of genes involved in cell cycle/proliferation and down-regulation of inhibitory/suppressive genes. CIML NK cells transduced with a chimeric antigen receptor (CAR) targeting the membrane-proximal domain of mesothelin (MSLN) further improved the antitumor responses against MSLN-expressing EOC cells and patient-derived xenograft tumor cells. CAR arming of the CIML NK cells subtanstially reduced their dysfunction in patient-derived ascites fluid with transcriptomic changes related to altered metabolism and tonic signaling as potential mechanisms. Lastly, the adoptive transfer of MSLN-CAR CIML NK cells demonstrated remarkable inhibition of tumor growth and prevented metastatic spread in xenograft mice, supporting their potential as an effective therapeutic strategy in EOC.