In recent years, cell-based immunotherapies have demonstrated promising results in the treatment of cancer. Chimeric antigen receptors (CARs) arm effector cells with a weapon for targeting tumor antigens, licensing engineered cells to recognize and kill cancer cells. The quality of the CAR-antigen interaction strongly depends on the selected tumor antigen and its expression density on cancer cells. CD19 CAR-engineered T cells approved by the Food and Drug Administration have been most frequently applied in the treatment of hematological malignancies. Clinical challenges in their application primarily include cytokine release syndrome, neurological symptoms, severe inflammatory responses, and/or other off-target effects most likely mediated by cytotoxic T cells. As a consequence, there remains a significant medical need for more potent technology platforms leveraging cell-based approaches with enhanced safety profiles. A promising population that has been advanced is the natural killer (NK) cell, which can also be engineered with CARs. NK cells which belong to the innate arm of the immune system recognize and kill virally infected cells as well as (stressed) cancer cells in a major histocompatibility complex I independent manner. NK cells play an important role in the host’s immune defense against cancer due to their specialized lytic mechanisms which include death receptor (i.e., Fas)/death receptor ligand (i.e., Fas ligand) and granzyme B/perforin-mediated apoptosis, and antibody-dependent cellular cytotoxicity, as well as their immunoregulatory potential via cytokine/chemokine release. To develop and implement a highly effective CAR NK cell-based therapy with low side effects, the following three principles which are specifically addressed in this review have to be considered: unique target selection, well-designed CAR, and optimized gene delivery.

Human transferrin protein (Tf) modified polyplexes have already displayed encouraging potential for receptor‐mediated nucleic acid delivery into tumors. The use of a blood‐derived targeting protein and polydisperse macromolecular cationic subunits however presents a practical challenge for pharmaceutical grade production. Here, Tf receptor (TfR) targeted small interfering RNA (siRNA) polyplexes are designed that are completely composed of synthetic, monodisperse, and sequence‐defined subunits generated by solid‐phase supported synthesis. An optimized cationizable lipo‐oligoaminoamide (lipo‐OAA) is used for siRNA core polyplex formation, and a retro‐enantio peptide (reTfR) attached via a monodisperse polyethylene glycol (PEG) spacer via click chemistry is applied for targeting. Improved gene silencing is demonstrated in TfR‐expressing KB and DU145 cells. Analogous plasmid DNA (pDNA) polyplexes are successfully used for receptor‐mediated gene delivery in TfR‐rich K562 cells and Neuro2a cells. Six lipo‐OAAs differing in their lipidic domain and redox‐sensitive attachment of lipid residues are tested in order to evaluate the impact of core polyplex stability on receptor‐dependent gene transfer.

For complexation of mRNA into polyplexes, double-pH-responsive lipo-xenopeptides (XP), comprising tetraethylene pentamino succinic acid (Stp) and lipoamino fatty acids (LAFs), were combined with PEGylated lipids, either DMG-PEG 2 kDa (DMG-PEG) or azido-group-containing DSPE-PEG 2 kDa (DSPE-PEG-N3), to increase colloidal stability and to facilitate ligand-mediated targeted mRNA delivery. LAF-XPs mixed with DMG-PEG at low (1.5% and 3%) molar ratios improved colloidal stability and retained transfection efficiency. PEGylation also enabled the formulation of otherwise unstable carrier complexes and prevented aggregation induced by salt, proteins, and serum. PEGylation of more positively charged Stp-LAF₂ mRNA polyplexes decreased fibrinogen adsorption. More neutral, LAF-rich Stp-LAF₄ polyplexes exhibited low fibrinogen binding without PEGylation. Intravenous administration of these stabilized mRNA complexes demonstrated enhanced biosafety while preserving transfection efficiency. DSPE-PEG-N3 was selected for cell targeting after strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated click-coupling of DBCO-modified ligands. Higher PEG ratios (10% and 20%) provided effective shielding but reduced transfection efficiency, a drawback known as the “PEG dilemma”. Functionalization with an EGFR-targeting ligand restored transfection in EGFR-positive cell lines in a ligand-specific manner. High transfection efficiency is consistent with a lipophilic-to-hydrophilic polarity switch of LAF-XP carriers upon endosomal protonation, triggering dissociation of the PEG lipids and deshielding of the polyplex.

An effective chimeric antigen receptor (CAR)‐based immunotherapy depends on both a suitable immune cell platform and a tumor‐specific antigen to overcome barriers in solid tumors. Natural killer (NK) cell lines are promising platforms for CAR constructs due to their inherent tumor‐killing ability, safety profile, and feasibility for standardized, off‐the‐shelf therapeutic use. Herein, four human NK cell lines (YT, KHYG1, NKL, and NK92) were retrovirally transduced with an anti‐Hsp70 CAR targeting membrane‐bound heat shock protein 70 (mHsp70), a tumor‐specific antigen with broad expression on many solid tumors, but not normal cells. Computational modeling suggested a strong binding between the CAR and the extracellular domain of mHsp70. Although all NK cell lines exhibited successful CAR integration and surface expression, only NKL and NK92 cells maintained stable CAR expression and long‐term viability. The anti‐Hsp70 CAR NKL and NK92 cells demonstrated enhanced expression of activation markers and secretion of cytotoxic effector molecules, and robust target‐specific killing of mHsp70‐positive cancer cells, while sparing mHsp70‐negative targets. Our findings validate the therapeutic potential of anti‐Hsp70 CAR NK cells and the suitability of NKL and NK92 cells for advancing off‐the‐shelf CAR NK cell therapies, thereby offering a promising strategy for targeting a broad range of solid tumors expressing mHsp70.

Synthetic polyinosinic:polycytidylic acid (poly(I:C)) offers an attractive cancer therapeutic by operating on two fronts at once, combining direct tumor cell killing with immunostimulatory activity. Yet, these dual functions can only be efficiently harnessed when intracellular delivery is sufficiently effective to enable poly(I:C) to reach and activate its intracellular receptors. We addressed this delivery challenge by developing pH-responsive formulations using lipoamino fatty acid xenopeptide (LAF-XP) carriers, composed of polar cationizable succinoyl tetraethylene pentamine (Stp) and apolar cationizable LAF building blocks in defined architectures. In particular, poly(I:C)-lipid nanoparticles (LNPs) formulated with bundle LAF₄-Stp₁ XP carriers displayed increased anti-tumoral activity at decreased dosage across multiple cancer cell models, compared to control formulations. In parallel, LAF-XP LNP-delivered poly(I:C) activated immune responses, including CXCL10 production by tumor cells, and activation of peripheral blood mononuclear cells (PBMCs), characterized by increased phenotypic markers (CD69 and LAMP-1/CD107a) and functional molecules (e.g., IFN-γ and granzyme B). Conditioned supernatant of pre-stimulated PBMCs with poly(I:C) reduced cancer cell viability, highlighting the contribution of PBMC-released factors to cancer cell death. Of particular novelty is the combination of poly(I:C) with siRNA-mediated survivin knockdown to increase apoptosis in cancer cells using the bundle LAF-XP LNP. Collectively, our findings establish efficient LAF-XP LNPs as a versatile platform that supports multi-layered therapeutic strategies.