Ex Vivo Production of Large Numbers of Genetically Modified NK Cells from Cord Blood or Mobilized Peripheral Blood CD34 + Cells Using Notch Ligand Delta-like 4 Culture System
Authors: Joshi Akshay , Andre Isabelle , Gaudeaux Pierre , Moirangthem Ranjita Devi , Soheili Tayebeh Shabi , Cavazzana Marina , Lagresle-Peyrou Chantal 
Published: November 2021

Abstract
Natural killer (NK) cells are an essential component of human innate immune, with a remarkable ability to provide protection against cancer and viral infections. NK-based immune therapy has several advantages over T cell immunotherapy: NK cells do not cause graft-versus-host disease and do not induce T-cell driven inflammatory cytokine storm. Moreover, unlike T cell, NK cells do not need prior sensitization, specific antigen recognition and clonal expansion for their cytotoxic effector functions and they can rapidly trigger cytotoxicity against their targets. NK cells can be expanded from multiple sources including peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), or mobilized peripheral blood (mPB) CD34 + cells. Yet the lack of efficient methods for in-vitro NK cell expansion, purification and genetic modification limits the clinical use of NK cell therapy.
We aimed at developing a simple, scalable and clinically feasible technique for therapeutic NK cell production based on our recently published culture system that can produce large numbers of unmodified or genetically modified CD7 + T lymphoid progenitors from UCB and mPB CD34 + cells in 7 days on immobilized Notch-ligand delta-like 4 recombinant protein DLL4. Since T cells and NK cells share a common T/NK cell progenitor, we are interested to investigate the NK cell potential of DLL4 culture-generated T lymphoid progenitors.
Firstly, we tested the NK cell potential of the transduced or non- transduced UCB or mPB CD34 +-derived progenitors from DLL4 culture by subjecting them to a feeder free NK cell differentiation for 3 weeks. HSPC-derived progenitors can be efficiently transduced with lentiviral vectors (average transduction efficacy: 50%). The transduced/ non-transduced progenitors were able to efficiently differentiate into NK (CD3 -CD56 +) cells beginning from one week of differentiation, reaching a frequencies of NK cells of &gt;90% without any detectable T cell contamination at week 2. The phenotypic characterization of the NK cells demonstrates the presence of activation receptors such as NKG2D, DNAM-1, NKp30, NKp44 and NKp46 while lacking the inhibitory receptors like KLRG1, KIR2DL2/DL3 and KIR3DL1/DL2. Interestingly, these cells express the transcription factors known to be essential for NK cell differentiation and functions such as Eomes, T-bet and ID2. Additionally, the CB or mPB HSPC-derived NK cells (both transduced/ non-transduced) express perforin and granzyme B reflecting their ability to show cytotoxic potential.
Further, the stimulation of UCB or mPB derived NK cells with myelogenous leukemia cell line K562 cells showed high level of degranulation, which is the key step for Interferon gamma (IFNg) induction. An analysis cytotoxic activity of the CB or mPB derived NK cells against K562 cells and monocytic leukemia cell line THP1 cells showed their ability to efficiently kill K562 cells compared to THP1 cells, however the UCB derived NK cells were highly cytotoxic compared to mPB derived NK cells.
These data suggests that our DLL4 culture system, along with feeder-free NK cell differentiation is a unique combination that is able to give rise to high number of pure NK cell population with high cytotoxic potential. These results lay a foundation towards an easier approach to NK cell therapy for effective treatment of cancers and viral infections, which will be developed in collaboration with Smart Immune Inc.

Disclosures
Cavazzana: Smart Immune: Other: co-founder.