Revolutionising Cancer Immunotherapy: Advancements and Prospects in Non-Viral CAR-NK Cell Engineering

Abstract

The recent advancements in cancer immunotherapy have spotlighted the potential of natural killer (NK) cells, particularly chimeric antigen receptor (CAR)-transduced NK cells. These cells, pivotal in innate immunity, offer a rapid and potent response against cancer cells and pathogens without the need for prior sensitization or recognition of peptide antigens. Although NK cell genetic modification is evolving, the viral transduction method continues to be inefficient and fraught with risks, often resulting in cytotoxic outcomes and the possibility of insertional mutagenesis. Consequently, there has been a surge in the development of non-viral transfection technologies to overcome these challenges in NK cell engineering. Non-viral approaches for CAR-NK cell generation are becoming increasingly essential. Cutting-edge techniques such as trogocytosis, electroporation, lipid nanoparticle (LNP) delivery, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing and transposons not only enhance the efficiency and safety of CAR-NK cell engineering but also open new avenues for novel therapeutic possibilities. Additionally, the infusion of technologies already successful in CAR T-cell therapy into the CAR-NK paradigm holds immense potential for further advancements. In this review, we present an overview of the potential of NK cells in cancer immunotherapies, as well as non-viral transfection technologies for engineering NK cells.

Keywords: chimeric antigen receptor; immunotherapy; natural killer cell; non‐viral transfection; tumour microenvironment.

FIGURE 1

The history of NK cell–based immunotherapies. Key discoveries, important clinical trials, and highlights that were prominent in the last decades are illustrated. NK: natural killer; LANAK: lymphokine‐activated natural killer; IL‐2: interleukin‐2; ACT: adoptive cell therapy; CAR: chimeric antigen receptor; CD: cluster of differentiation; PTX: paclitaxel; cMLVs: cross‐linked multilamellar liposomal vesicles.

FIGURE 2

Illustration of different types of non‐viral technologies for NK cell engineering. (A). Trogocytosis: A process by which NK cells acquire membrane patches from APCs. The CAR is transferred from the APC to the NK cell, which enables functional modification. (B). CRISPR‐Cas9: A genome editing technique where the Cas9 protein, guided by sgRNA, introduces double‐stranded breaks in DNA. Donor DNA is subsequently integrated during the repair process, allowing for precise genomic insertion. (C). Electroporation: The transient formation of pores in cell membranes via electrical pulses facilitates the entry of CAR DNA and CAR mRNA directly into the cytoplasm. (D). Transposons: A transposase‐mediated ‘cut‐and‐paste’ mechanism, mobilised by transposase (Tnpase) molecules, can integrate a GOI into the host genome. The transposase binds to the TIRs, induces double‐stranded breaks, and excises the mobile element from the donor DNA leaving behind a footprint. The transposon–transposase complex finds a suitable TS and performs integration, producing a TSD. (E). LNP: LNP encapsulates RNA and facilitates its delivery into the cell, leveraging the cell's endocytic pathways for subsequent expression. APC: antigen‐presenting cell; sgRNA: single‐guide RNA; GOI: gene of interest; TIRs: terminal inverted repeats; TS: target site; TSD: target site duplication; LNP: lipid nanoparticle.

FIGURE 3

Graphical illustration of the challenges and potential enhancements associated with CAR‐NK cell therapy via trogocytosis. The upper segment illustrates the limitations of trogocytosis, highlighting issues such as fratricide, enhanced by trogocytosis‐mediated transfer of tumour cell antigens to NK cells, NK cell exhaustion marked by exhaustion markers, and antigen loss leading to the inability of antibody–antigen binding. The lower segment proposes potential strategies to augment the efficacy of the therapy, including a dual‐CAR strategy, pharmacological targeting utilising TAK981 to boost CH25H expression and modifications to signalling domains. CAR: chimeric antigen receptor; NK: natural killer; AI‐CAR: both activating chimeric antigen receptor and an inhibitory chimeric antigen receptor; CH25H: cholesterol 25‐hydroxylase; CD: cluster of differentiation.

FIGURE 4

Mechanism of promising technologies in non‐viral delivery. (A). Sonoporation. Microbubbles expand or contract when subjected to US, which create temporary openings in cellular membrane, allowing an influx of external cargo molecules into the cytosol. (B). Graphical illustration of VNB photoporation. VNB–induced photoporation involves pretreating cells with photothermal agents, commonly gold nanoparticles, which attach to their exteriors, followed by pulsed laser exposure. The rapid expansion and subsequent collapse of these bubbles momentarily disrupt the cellular membrane, creating openings through which macromolecules, like mRNA, can enter the cytoplasm. (C). The figure illustrates nanowire‐mediated cargo delivery into the cell. Solid nanowires create pore formation in the cell membrane, allowing for the passive diffusion of extracellular cargo directly into the cytoplasm. (D). The figure illustrates hollow nanoneedles delivering for cargo into a cell by first creating membrane pores, and then pumping the cargo directly into the cytoplasm. US: ultrasound stimulation; VNB: vapour nanobubble.