Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

A novel CD34-derived hinge for rapid and efficient detection and enrichment of CAR T cells.

Immunotherapy including chimeric antigen receptor (CAR) T cell therapy has revolutionized modern cancer therapy and has achieved remarkable remission and survival rates for several malignancies with historically dismal outcomes. The hinge of the CAR connects the antigen binding to the transmembrane domain and can be exploited to confer features to CAR T cells including additional stimulation, targeted elimination or detection and enrichment of the genetically modified cells. For establishing a novel hinge derived from human CD34, we systematically tested CD34 fragments of different lengths, all containing the binding site of the QBend-10 monoclonal antibody, in a FMC63-based CD19 CAR lentiviral construct. A final construct of 99 amino acids called C6 proved to be the best candidate for flow cytometry-based detection of CAR T cells and >95% enrichment of genetically modified T cells on MACS columns. The C6 hinge was functionally indistinguishable from the commonly used CD8α hinge in vitro as well as in in vivo experiments in NSG mice. We also showed that the C6 hinge can be used for a variety of different CARs and mediates high killing efficacy without unspecific activation by target antigen-negative cells, thus making C6 ideally suited as a universal hinge for CARs for clinical applications.

Novel DRAQ5™/SYTOX® Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk.

BACKGROUND: Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. METHODS: Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX® blue dye as well as antibodies to characterize cells within the pellet fraction. RESULTS: Flow cytometry analysis identified viable DRAQ5™+ /SYTOX® Blue- cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. CONCLUSIONS: Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content. © 2018 International Clinical Cytometry Society.

MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITF(low)/c-Jun(high) syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITF(low)/c-Jun(high) melanomas to counteract their growth-promoting and immunosuppressive functions.