Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy

Abstract

Due to their efficient recognition and lysis of malignant cells, natural killer (NK) cells are considered as specialized immune cells that can be genetically modified to obtain capable effector cells for adoptive cellular treatment of cancer patients. However, biological and technical hurdles related to gene delivery into NK cells have dramatically restrained progress. Recent technological advancements, including improved cell expansion techniques, chimeric antigen receptors (CAR), CRISPR/Cas9 gene editing and enhanced viral transduction and electroporation, have endowed comprehensive generation and characterization of genetically modified NK cells. These promising developments assist scientists and physicians to design better applications of NK cells in clinical therapy. Notably, redirecting NK cells using CARs holds important promise for cancer immunotherapy. Various preclinical and a limited number of clinical studies using CAR-NK cells show promising results: efficient elimination of target cells without side effects, such as cytokine release syndrome and neurotoxicity which are seen in CAR-T therapies. In this review, we focus on the details of CAR-NK technology, including the design of efficient and safe CAR constructs and associated NK cell engineering techniques: the vehicles to deliver the CAR-containing transgene, detection methods for CARs, as well as NK cell sources and NK cell expansion. We summarize the current CAR-NK cell literature and include valuable lessons learned from the CAR-T cell field. This review also provides an outlook on how these approaches may transform current clinical products and protocols for cancer treatment.

The emerging role of off-the-shelf engineered natural killer cells in targeted cancer immunotherapy

“…The co-activating proteins are usually based on the CD28 family (CD28 or ICOS), the TNF receptor family (4-1BB, OX40, or CD27), or the signaling lymphocytic activation molecule (SLAM)-related receptor family (2B4). 67 , 71 A study comparing ErbB2-targeted NK cells expressing CD3ζ alone, CD28/CD3ζ, or CD137/CD3ζ found that the second-generation constructs displayed increased cytotoxicity compared to the first-generation CAR. 72 …”

“…The main gene modification methods utilized to generate CAR NK cells are viral transduction and transfection. 58 , 71 Retrovirus and lentivirus-based vectors have been widely applied in the production of CAR NK cells due to stable gene integration and high transduction rates, especially in blood-derived NK cells. Transduction levels of up to 60% were achieved in peripheral blood NK cells using retroviral vectors, 73 while a 73% transduction efficacy was achieved using lentiviral vectors in NK cells derived from cord blood.…”

New Orders to an Old Soldier: Optimizing NK Cells for Adoptive Immunotherapy in Hematology

“…Sutlu et al stimulated 9.8 × 10 9 NK cells derived from peripheral blood in a closed, automated, sterile, large-scale bioreactor consisting of a bag on a heated rocking or static platform under feeder-free GMP conditions after 21 days [ 90 ]. Miltenly’s Prodigy, Lonza’s Wave and G-Rex static bioreactors are automated and closed expansion equipments that have facilitated the manufacture of GMP-grade products [ 5 ]. Although large-scale generation of GMP-grade products is possible using serum-free commercial media such as AIMV media (Life Technologies, USA), X-VIVO media (BioWhittaker, Belgium) and SCGM media (CellGenix, Germany) [ 91 ], there are no standard manufacturing techniques because of the variation of culture conditions throughout clinical trials.…”

“…Some companies have already adapted electroporation in closed GMP-compliant production systems. Sleeping beauty transposon and CRISPR/Cas9-specific locus knock-in strategies need to be explored further for enhanced transduction and longer persistence in vivo [ 5 ].…”

“…Several activating NK cell receptors and co-stimulatory molecules recognize tumors [ 4 ]. NK cells also exhibit antigen-dependent cellular cytotoxicity (ADCC) by detecting antibodies on tumor cells through the low-affinity Fcγ CD16 receptor [ 5 ].…”