Genetic Engineering of Natural Killer Cells
المؤلف:
Hoffman, R., Benz, E. J., Silberstein, L. E., Heslop, H., Weitz, J., & Salama, M. E.
المصدر:
Hematology : Basic Principles and Practice
الجزء والصفحة:
8th E , P229
2025-12-15
117
NK cells can be genetically modified to express new proteins that augment their ability to eliminate target cells and then infused for therapeutic purposes. The use of HLA-mismatched NK cells provides for an expanded menu of cell types that can serve as starting mate rial. Strategies that have been employed include the expression of (1) modified activating receptors, (2) machinery to downregulate inhibitory receptors, (3) soluble or membrane-bound cytokine, and (4) chimeric activating receptors (CARs). Genetically modified NK cells may then be infused into cancer patients following lymphodepleting chemotherapy.
Based on the clinical success of CAR-T-cell therapy for CD19 expressing lymphomas there has been considerable interest in generating analogous NK cell products or so-called CAR-NK. This approach has several theoretical advantages over CAR-T-cell therapies. While CAR-T-cell therapy requires genetic manipulation of autologous or HLA-matched T cells, CAR-NK may be generated from multiple different cell sources, and as is the case with HCT, HLA mismatches can be beneficial to the NK cell therapeutic effect. Reduced expression of MHC class I molecules or target antigen severely limits the cytotoxic capacity of infused CAR-T cells and represents an important mechanism of resistance to this form of cell therapy. In contrast, NK cells possess an innate ability to detect and eliminate such target cells due to the ability of NK cells to detect activating/stress ligands and missing self. Although NK cells have the ability to conduct serial killing, infused NK cells have a limited lifespan and have not been known to cause significant side effects or GVHD. On the other hand, CAR-T-cell therapy can result in serious and sometimes fatal toxicities including cytokine release syndrome and neurotoxicity in the form of immune effector cell-associated neurologic syndrome (ICANS). It is not always possible to obtain sufficient numbers of high-quality T cells from chemotherapy-refractory patients. Also, the manufacturing process can take 2 to 4 weeks and patients must have therapy held for at least 2 weeks prior to apheresis. Thus, the timely administration of CAR-T cells can be problematic in patients who have proven refractory to standard therapies. CAR-T cell therapy requires post-treatment hospitalization for several weeks. In contrast, it is possible to envision “off-the-shelf” CAR-NK cell products being administered in the outpatient setting as a bridging therapy to patients experiencing recurrent disease.
Advances made in the field of CAR-T-cell therapy have been applied to the development of CAR-NK cells. The CAR structure usually consists of an extracellular scFv derived from an antibody against a tumor antigen, a hinge region connecting this antigen-recognition sequence to a transmembrane domain (TM), and one or more intracellular signaling domains. The scFv consists of a heavy and a light variable fragment (VH, VL) joined by a flexible linker. The binding affinities and order of the variable fragments as well as the length of the linker can impact CAR affinity for an antigen. The hinge sequence is typically based on known proteins with similar structure (CD8alpha, CD28, CH2/3 domains), and its length can be adjusted to optimize antigen binding. The TM domain is a hydro phobic region that crosses the cell membrane and serves to anchor the CAR. The choice of TM is important since some sequences can pro mote activation-induced cell death or alter CAR cell surface distribution. First-generation CAR-T constructs contained only a CD3zeta activating domain. However, subsequent generations included one or more additional co-stimulatory domains derived from either the TNF receptor family (4-1BB, CD27, OX40) or the CD28 family. CAR-NK constructs may contain NK-specific activating domains (e.g., DAP10 or DAP12) in place of or alongside CD3zeta. Co-stimulatory domains derived from 2B4 and CD137 have also been evaluated.
As with NK cell infusions for leukemia, there are multiple cell types that can be used for the generation of CAR-NK cells. Peripheral blood CD56+ NK cells can be obtained through apheresis, depleted of T and B cells, and expanded in a stimulatory environment. These NK cells possess a mature phenotype and effectively eliminate tumor cells but exhibit low proliferation. Lymphocytes derived from umbilical cord blood have a high proportion of NK cells of an immature phenotype that exhibit reduced expression of CD16, perforin, and granzyme B. However, their proliferative capacity is much greater than that of NK cells derived from peripheral blood. iPST-derived CAR-NK cells exhibit a high proliferative capacity but have low levels of CD16 and KIR and high levels of inhibitory NKG2A. These deficits can be rectified via insertion of missing cytotoxic components. Also, the NK-92 cell line can be genetically modified to express a CAR molecule and then infused following irradiation. Low expression of KIR and CD16 by this cell line is a limiting factor.
NK cells are notoriously resistant to viral transduction with retrovirus- or lentivirus-based constructs. The use of modified viral vectors, cationic transfection reagents, and cytokine activation can help improve the transduction efficiency. Electroporation can result in a high rate of gene uptake; however, stable integration of the construct is not possible and consequently there is transient expression of the CAR construct. The use of transposon technology and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems in conjunction with electroporation or viral approaches can improve the efficiency of gene uptake and dictate the site of integration.
Expansion of the NK cell product pre-infusion can be achieved with various combinations of cytokines, and these can be chosen with the goal of directing the NK cell population toward a particular phenotype, such as the use of IL-12, IL-15, and IL-18 to induce a memory NK cell. However, if high concentrations of these factors are used there is the potential for cytokine addiction and reduced in vivo efficacy. Genetically engineered feeder cells represent an alternate method for driving the expansion of NK cells. NK cells have a limited ability to proliferate, and unlike T cells, infused NK cells cannot be detected in a patient’s circulation past 1 to 2 weeks post-transfusion. For this reason, the systemic administration of IL-2 or IL-15 has been considered as a means of lengthening the time frame of CAR-NK cell activity. The delivery of CAR-NK constructs in combination with anti-tumor mAbs, checkpoint inhibitors, and targeted drugs to eliminate immune suppressor cells is being investigated.
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