Delivery Technologies to Engineer Natural Killer Cells for Cancer Immunotherapy

Purpose: CAR T cell therapies have achieved clinical success but face limitations including restriction to autologous cell sources (to avoid HLA mismatch and GvHD), cumbersome manufacturing, high costs, and poor efficacy in solid tumours. NK cells have emerged as promising alternatives because they can kill cancer cells without HLA matching, can be derived from allogeneic sources, and have the potential to become "off-the-shelf" therapeutics. However, NK cells are resistant to genetic engineering and have limited proliferation and persistence, creating a need for improved delivery technologies to maximize their therapeutic potential. ---

Hypothesis: The review’s central thesis is: if advanced delivery technologies—including viral, non-viral (electroporation, trogocytosis, CRISPR-Cas9, CARTs, LNPs), and surface engineering approaches—are applied to NK cells, then NK cells can be effectively engineered for cancer immunotherapy with improved persistence, target specificity, and potential for allogeneic/off-the-shelf applications. ---

Aims: - Provide an overview of NK cell biology and their use in cancer immunotherapy. - Review current clinical advancements of engineered NK cells for treating haematological and solid tumours. - Discuss various NK cell sources for immunotherapy (NK-92, PB-NK, CB-NK, iNK). - Review viral, non-viral, and nanoparticle-based strategies for engineering NK cells. - Discuss surface engineering strategies (TRAIL-liposomes, glycoengineering, aptamer engineering). - Provide perspective on future directions for engineered NK cell therapies. ---

Delivery system: NK cell sources: - NK-92 cell line (immortalized, readily available, lacks KIR, low CD16) - Peripheral blood NK (PB-NK) cells (CD56^dim CD16^+, highly cytotoxic, require extensive ex vivo expansion) - Cord blood NK (CB-NK) cells (less mature, higher proliferation, lower GvHD risk) - Induced pluripotent stem cell-derived NK (iNK) cells (unlimited proliferative potential, enhanced transfection efficiency, low CD16, poor persistence) Genetic engineering — viral vectors: - Retroviral vectors (IL-2 cDNA, CAR constructs); efficiency 60–90% for NK-92, ~50% for primary NK - Lentiviral vectors (do not require dividing cells); require cationic reagents (polybrene, protamine sulfate, DEAE-dextran, poly-L-lysine, Retronectin) Genetic engineering — non-viral vectors: - Electroporation/Nucleofection (mRNA or plasmid DNA; 80–90% transfection with mRNA) - Trogocytosis-mediated methods (co-culture with K562 "donor" cells expressing CCR7 or CAR) - CRISPR-Cas9 (targeted knockout of ADAM17, PDCD1; ~90% modification efficiency) - Charge-altering releasable transporters (CARTs; mRNA delivery) - Lipid nanoparticles (LNPs; ionizable lipid, cholesterol, helper phospholipid, PEG-lipid; mRNA delivery) Surface engineering: - TRAIL-based liposomes (functionalized with TRAIL and anti-NK1.1) - Glycoengineering (CD22 ligands, E-selectin) - Aptamer engineering (ApEn-NK cells; CD30-specific aptamers) Payloads: CAR constructs, IL-2 cDNA, IL-15, mRNA, plasmid DNA, CRISPR-Cas9 components (Cas9 mRNA, sgRNA, RNPs), paclitaxel, suicide genes (iCasp9). ---

Approach: Narrative review of preclinical and clinical literature. No primary experimental groups. Model systems discussed include: - In vitro: NK-92, primary NK cells, PB-NK, CB-NK, iNK cells, various cancer cell lines. - In vivo: Mouse models of leukaemia, lymphoma, breast cancer, glioblastoma, ovarian cancer, and other solid tumours. - Clinical trials: Phase I/II trials of CAR-NK cells (Table 1 lists trial numbers including NCT02742727, NCT02892695, NCT02944162, NCT03383978, NCT03940820, NCT03056339, NCT04245722, etc.). - Disease context: Haematological malignancies (leukaemia, lymphoma, multiple myeloma) and solid tumours (glioblastoma, breast, ovarian, prostate, non-small cell lung cancer). ---

Key methods: Techniques and endpoints highlighted across cited studies: - Flow cytometry for CAR expression, NK cell phenotype, CD16 expression, cytokine production. - In vitro cytotoxicity assays (target cell killing, IFN-γ secretion). - In vivo tumour growth inhibition and survival analysis. - Transfection efficiency and cell viability assays. - CRISPR-Cas9 editing efficiency (indel analysis). - Clinical response assessment (complete remission, overall response rate, GvHD/CRS incidence). - Trogocytosis-mediated protein transfer quantification. ---

Key results: - CAR T cell dominance: ~96.4% of 520 active CAR-based cell therapy trials globally are CAR T-cell-based; CAR-NK trials represent a very small fraction. - Retroviral transduction efficiency: 60–90% for NK-92 cells; ~50% for primary NK cells. - Electroporation: mRNA electroporation achieved 80–90% transfection in resting and ex vivo expanded human NK cells; median cell viability ~90%; HER2-CAR-NK-92 cells retained 60–90% viability with significant tumour growth inhibition in a mouse breast cancer model. - CRISPR-Cas9: Pomeroy et al. efficiently modified ~90% of PB-NK cells with ADAM17 and PDCD1 knockout, with increased cytokine production and tumour cytotoxicity. - CB-NK clinical trial (NCT03056339): Liu et al. reported 73% response rate (8/11 patients) and 7 complete remissions in non-Hodgkin's lymphoma or CLL; no GvHD, CRS, or neurotoxicity. - Trogocytosis: 80% of NK cells expressed CCR7 after 1 h co-culture with K562 cells; 18.6% expressed anti-CD19-CAR after 1 h; CCR7 and CD19 expression ceased after 72 h and 2 h, respectively. - CARTs: CART-mediated mRNA transfection was as efficient as high-dose electroporation but with higher overall cell viability; minimally altered NK cell phenotype and proteomic expression. - Glycoengineering: NK-92 cells modified with CD22 ligands showed enhanced targeting and lysis of CD22-positive cancer cells. - Aptamer engineering: CD30-specific ApEn-NK cells showed enhanced targeting specificity and killing of CD30+ T-cell lymphoma. - Super NK cells: TRAIL and anti-NK1.1 protein-coated liposomes enhanced therapeutic potential in tumour-draining lymph nodes and prevented metastasis. ---

Interpretation: The authors conclude that genetic and surface engineering strategies for redirecting or boosting NK cell cytotoxicity are rapidly expanding. CAR-NK cells represent the majority of investigated NK cell-based approaches in clinical trials. With rapid advancements in drug delivery and gene-editing systems—particularly CRISPR-Cas9 and nanoparticle delivery—NK cell-based cancer immunotherapies could advance significantly in the near future. However, further research is essential to improve in vivo persistence, solid tumour efficacy, and manufacturing scalability for widespread clinical translation. ---

Limitations: - NK cell persistence: Poor proliferative potential and persistence in vivo, typically lasting only 1–2 weeks post adoptive cell transfer. - Genetic engineering resistance: NK cells are resistant to genetic engineering and undergo limited cell divisions. - Viral vector limitations: Risks of insertional mutagenesis, high manufacturing complexity/cost, and need for large quantities of virus. - Electroporation limitations: Potential for cell death and irreversible membrane damage. - Trogocytosis limitations: Rapid loss of acquired protein (CCR7 lost by 72 h; CD19 lost by 2 h). - CRISPR-Cas9 limitations: Off-target effects, immunogenicity, and in vivo delivery challenges. - Nanoparticle limitations: Not yet thoroughly explored in NK cells; potential liver/spleen accumulation and toxicity. - Solid tumour challenges: Complex and immunosuppressive TME limits NK cell efficacy; therapeutic targets with broad tumour coverage are needed. - Manufacturing: Scale-up, cost, and accessibility remain barriers for broad clinical implementation. - As a review: Not a systematic review or meta-analysis; no primary data. This response is AI-generated, for reference only.