CAR-NK cells for cancer immunotherapy: from bench to bedside | Biomarker Research | Full Text
nk cell lines
In general, considering the difficulty in isolating, purifying, and expanding primary nk cells, as well as the inefficiency in transduction of automotive constructs, well-established nk cell lines with indefinite expandability have been used in the clinical practice [25]. in particular, the representative il-2-dependent cell line nk-92 and the nk-92mi derivation exhibit splendid advantages of easy expansion, culture, and activation in the context of lymphocyte depletion, along with reliable and sustainable cytotoxicity after infusion against leukemic cells (fig. 1) [24]. For example, Boyiadzis and colleagues conducted a phase 1 clinical trial of NK92 cell-based adoptive immunotherapy in patients with relapsed and refractory acute myeloid leukemia (AML) and confirmed the feasibility, safety, and strong antileukemic activity [26]. simultaneously, it should be noted that nk92 cells originate from patients with non-hodgkin lymphoma and therefore require irradiation prior to infusion to eliminate the risks of malignant transformation and accompanying chromosomal abnormalities [27]. Another preclinical study on AML immunotherapy by Kloess et al verified that modified cd123-car-nk-92 cells showed higher levels of granzyme and interleukin secretion and preferable cytotoxic activities than primary cd123-car-nk cells derived from human donors. , while they revealed significant side-effect levels against non-malignant cells as well [28]. In addition, Binyamin et al. found that nk-92 cells revealed enhanced adcc when blocking inhibitory receptors (eg, kir2dl1, kir3dl1) and combined with rituximab [29].
Another classical nk yt cell line with psma transduction of prostate cancer cell antigen and deletion of shp-2 (ptpn11) has been reported with enhanced cytotoxicity [30]. meanwhile, the yt leukemic cell line has also demonstrated spontaneous cytotoxicity against b-lymphoma and a specific lytic effect on aml by targeting cd80+/cd86+ b-lymphoblastoid cells and cd33+ leukemia cells, respectively [31, 32]. Despite increasing references to car-NK cell-based cancer immunotherapy, most current studies are preclinical. however, observations from existing studies support new treatment concepts using car-nk cell lines with potent degranulation and selective cytotoxicity in malignancies [33].
Reading: Car nk review
nk cells (pb-nk) derived from peripheral blood
in the peripheral blood, nk cells account for a proportion of 5-20% of leukocytes, which are divided into the dominant subset cd56dimcd16high (85-95%) and the minimal subset cd56brightcd16low/neg (5-15%) [5, 34]. in addition, bp-nk cells express a wide range of active receptors and therefore have potential as splendid sources for the adoptive generation of car-nk cells (fig. 1) [21, 35]. In general, resting pb-nk cells reveal a tremendous cd3-cd56bright proliferative cell phenotype and are capable of secreting immunomodulatory cytokines, whereas the cd3-cd56dim counterpart possesses high cytotoxicity and poor proliferation in response to cytokine stimulation ( for example, il-2) [ 36].
to date, bp-nk cells and car-pb-nk cells derived from autologous and allogeneic donors have been most effective in the treatment of acute leukemia (clinically effective doses ranging from 1×106/kg to 9.3 × 106/kg ) while with relatively minimal activity against solid tumors [37,38,39]. recently, a preclinical study by quintarelli et al confirmed that cd19-car transduced pb-nk cells were sufficient to mediate robust cytotoxicity against b-cell precursor acute lymphocytic leukemia (bcp-all) and maintain function of all “native” nk coreceptors after genetic modification [40].
nk (uc-nk) cells derived from umbilical cord blood
unlike pb-nk cells, umbilical cord blood nk cells (uc-nk) only account for about 5% of total mononuclear cells (tncs), but offer unique alloreactive advantages for adoptive immunotherapy and increase the potential as a third-party product for broad clinical scalability (Fig. 1) [36, 41]. Despite the higher percentage of naïve NK cell population in circulating cord blood, the majority of UC-NK cells were adequate for differentiation into functionally mature and active effector cells and thus the successful acquisition of functional competence. after ex vivo costimulation with cytokine cocktails (eg, il-2, il-7, il-12, il-15, il-18) [36, 42]. For example, Xing et al reported that the low cytolytic activity of resident UC-NK cells in vivo is attributed to impaired lytic immunological synapse formation, as well as enhancement by IL-2 stimulation during ex-expansion and activation. alive [43]. Collectively, the existing literature indicates that uc-nk cells are phenotypically and functionally immature but capable of maturation [42].
It should be noted that an increasing number of investigators have turned to uc-nk cells to generate clinically or preclinically proven car-nk cells [44]. For example, a first-in-human Phase 1/2 clinical trial identified the viability of lymphocyte-depleted CAR-UC-NK cells for the treatment of relapsed and refractory CD19+ B-cell lymphoma, including seven cases with unexplained complete remissions. important toxic effects. effects [45]. Despite the once reported generation of more than 100 doses of engineered car-nk cells from one unit of cord blood, uc-nk cells have notable limitations in cell mass for large-scale adoptive immunotherapy but with a high level of expression of the nkg2a inhibitory receptor and poor in vitro cytotoxicity [36, 42, 46].
nk (p-nk) cells derived from placental blood
Despite the rare p-nk cell content (< 2%) in tncs, placental blood is more abundant compared to the aforementioned adult peripheral blood and umbilical cord blood (fig. 1). meanwhile, current strategies have indicated highly efficient generation of clinical grade cd3-cd56+ nk cells (an average of nearly 1 billion nk cells per donor) with markedly increased antitumor cytolytic activity from placental perfusion [38 ]. Compared to uc-nk cells, the derived p-nk cells are largely similar to uc-nk cells phenotypic and functionally, but display distinct microRNA expression profiles, immunophenotypes, and superiority in killing a wide range of cell lines. cancerous in vitro and therefore have potential. for car-p-nk cell-based immunotherapeutic development [38, 47]. in particular, guo et al recently raised the possibility of ameliorating p-nk cell cytotoxicity through crispr/cas9-induced cblb ablation [48].
stem cell-derived nk cells
now, prospective studies have also indicated the feasibility of deriving mature nk cells from cd34+ hematopoietic stem/progenitor cells (hspcs). In a preclinical study on the AML xenograft model, Cany et al reported the proof-of-concept safety and efficiency of targeting bone marrow-residing leukemic cells via the ccr6/ccl20 and cxcr3/cxcl10-11 axes in mice. nod/scid/il2rgnull [ 49].
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Human pluripotent stem cells (hpscs), including human induced pluripotent stem cells (hipscs) and human embryonic stem cells (hescs), possess potential for self-renewal and multilineage differentiation [50,51,52]. Over the past few decades, we and other investigators have reported the generation of progenitor cells and functional cells from HPSCs, including mesenchymal stromal/stem cells (MSCs), megakaryocytes (MKs), and NK cells [50, 53]. In particular, they differ from other cell sources with dominant limitations on NK cell survival and proliferation, HPSC-NK cells can be manufactured from the standardized HPSC population and thus meet clinical demands for cell products. large-scale homogeneous car-nk (fig. 1) [54, 55]. meanwhile, considering the relatively low efficiency of car transduction in primary nk cells, the deficiency of adult pb-nk cells and perinatal uc-nk cells and p-nk cells in cell activity against solid tumors could be overcome by car -hpsc genetically modified -nk cells through viral or non-viral strategies as hypothesized by pioneer researchers [54, 56].
In addition, it is considered that autos can be easily and conveniently delivered into hpscs using the non-viral transgenic approach [57, 58]. For example, Ni and his colleagues reported integration of the chimeric cd4ζ receptor into hpsc-derived nk cells (cd4ζ-hesc-nk cells, cd4ζ-hipsc-nk cells) with improved efficacy against human immunodeficiency virus ( hiv) / AIDS [57]. of note, li et al recently highlighted the feasibility of transducing car constructs with conventional nk cell-specific intracellular activation domains into ipsc-nk cells (car+ ipsc-nk cells) to further optimize tumor-specific recognition and cytotoxicity [ 59].
memory-like nk (ml-nk) cells
cytokine-induced ml-nk cells with dominant nkg2a checkpoint expression, phenotypically distinct from conventional nk cells in vivo, have been found to be safe and sufficient to induce remissions in aml patients, which are recognized as novel pathways to facilitate the -nk cell therapeutics (fig. 1) [60, 61]. therefore, we and other investigators assume that car-ml-nk cells possess a more potent and flexible response to a variety of cancer cell-associated triggers compared to conventional nk cells [62, 63]. It is noteworthy that differentiated memory-type car-NK cells showed elevated activation receptors against myeloid leukemia and prolonged survival in vivo regardless of typical kir-kir ligand interactions [61, 64]. meanwhile, numerous preclinical data have demonstrated superior degranulation and ifn-γ associated response of car-nk cells, as well as enhanced cancer cell killing and adcc effect against tumor cells [65, 66].
in particular, peripheral blood-derived ml-nks with truncated cd-19-car transduction were phenotypically and functionally mature and manifested significantly elevated ifn-γ secretion and degranulation, expanded recognition, and specifically cleared lymphoma resistant to nk compared to conventional car-nk cells or non-specific nk cells [63]. Furthermore, Foltz et al reported that car-NK cells could even survive and persist in vivo for more than 2 months following adoptive transfer in the immunocompatible environment, which would be a significant improvement on the short lifespan of conventional NK cells.
