Efficient Lentiviral Transduction of Genetically Barcoded AntiCD20-CAR-IL15 Cassette into Peripheral Blood-Derived Rhesus Macaque NK Cells Using Poloxamer 407 and CAR-NK In Vitro and In Vivo Effects

Authors

• TB Hayal
• C Wu
• AA Mulla
• DSJ Allan
• BB Duncan
• SG Hong
• R Basar
• K Rezvani
• CE Dunbar

Abstract

Natural killer (NK) cells are pivotal in immune surveillance and antitumor responses by detecting and eliminating infected or cancerous cells. Their potential for cell-based therapies, particularly through chimeric antigen receptor (CAR) technology, has been underscored in recent trials, emphasizing low graft-versus-host disease risk and strong killing abilities. Efforts are concentrated on enhancing NK cell killing potency, persistence, and specificity via genetic modifications. While VSV-G pseudotyped lentiviral vectors have been extensively used in engineering T cells for clinical applications, they have been limited by low transduction efficiency of NK cells. Baboon endogenous virus envelope lentiviruses offer an alternative but have limitations in terms of titer and clinical development. Improving methods for efficient NK cell modification is critical for advancing CAR-NK cell therapies, particularly in pre-clinical models like the rhesus macaque (RM).
Our study aimed to optimize lentiviral transduction of RM-NK cells using Poloxamer 407 (P407). NK cells were expanded ex vivo using IL-2 and uAPC feeders before lentiviral transduction, resulting in 40-60% of stable transduction and effective expression of antiCD20-CAR and RM-IL-15. IL15-armored CAR-NK cells demonstrated enhanced cytotoxicity against CD20-positive tumor cells in vitro and showed improved persistence and reduced tumor burden in xenograft models compared to unarmored CAR-NK cells.
These findings underscore the efficacy of P407 in enhancing lentiviral transduction of RM NK cells and the therapeutic potential of IL15-armored CAR-NK cells. Ongoing research involves further characterization of these cells' clonal dynamics and functional attributes in autologous RM models, aiming to advance their clinical applicability.

Scientific Focus Area: Immunology

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