Functional assessment for clinical use of serum-free adapted NK-92 cells

Natural killer (NK) cells stand out as promising candidates for cellular immunotherapy due to their capacity to kill malignant cells. However, the therapeutic use of NK cells is often dependent on cell expansion and activation with considerable amounts of serum and exogenous cytokines. We aimed to develop an expansion protocol for NK-92 cells in an effort to generate a cost-efficient, xeno-free, clinical grade manufactured master cell line for therapeutic applications. By making functional assays with NK-92 cells cultured under serum-free conditions (NK-92(SF)) and comparing to serum-supplemented NK-92 cells (NK-92(S)) we did not observe significant alterations in the viability, proliferation, receptor expression levels, or in perforin and granzyme levels. Interestingly, even though NK-92(SF) cells displayed decreased degranulation and cytotoxicity against tumor cells in vitro, the degranulation capacity was recovered after overnight incubation with 20% serum in the medium. Moreover, lentiviral vector-based genetic modification efficiency of NK-92(SF) cells was comparable with NK-92(S) cells. The application of similar strategies can be useful in reducing the costs of manufacturing cells for clinical use and can help us understand and implement strategies towards chemically defined expansion and genetic modification protocols

Plasmonic selection of ssDNA aptamers against fibroblast growth factor receptor

In this work, we describe the selection of ssDNA aptamers targeting fibroblast growth factor receptor binding protein 3 K650E, which has roles in cell division, growth, and differentiation through the kinase cascade. The selection process was based on the label-free, real-time monitoring of binding interactions by surface plasmon resonance, allowing for convenient manipulation of the selection rounds. Next generation sequencing data provided four major motif families from which nine individual sequences were selected based on their abundance levels. Electrophoretic mobility shift assays revealed binding of the selected aptamers to the target protein without significant interference from fibroblast growth factor receptor binding protein 2, indicating the selectivity of the aptamers. The dissociation constant at equilibrium for the best aptamer candidate, SU-3, was found to be (28.2 ± 19.6) × 10–9 M (n = 5) using a single-cycle kinetic analysis method. Advantages of the experimental setup and potential applications of the selected aptamers are discussed.Sabanci Universit

Enhancing Functional Maturation of iPSC-derived Endodermal Hepatic Organoid through Modified Culture Conditions

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Engineering antigen‐specific NK cell lines against the melanoma‐associated antigen tyrosinase via TCR gene transfer

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T‐lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL‐TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off‐target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen‐specific TCR complex on NK cell lines NK‐92 and YTS, demonstrated by using a TCR against the HLA‐A2‐restricted tyrosinase‐derived melanoma epitope, Tyr368‐377. Most importantly, the introduction of a TCR complex to NK cell lines enables MHC‐restricted, antigen‐specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of “mispairing” in TCR gene therapy