Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient’s blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.

Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies.

The Wharton's jelly of the umbilical cord is rich in mesenchymal stem cells (UC-MSCs) that fulfill the criteria for MSCs. Here we describe a novel, simple method of obtaining and cryopreserving UC-MSCs by extracting the Wharton's jelly from a small piece of cord, followed by mincing the tissue and cryopreserving it in autologous cord plasma to prevent exposure to allogeneic or animal serum. This direct freezing of cord microparticles without previous culture expansion allows the processing and freezing of umbilical cord blood (UCB) and UC-MSCs from the same individual on the same day on arrival in the laboratory. UC-MSCs produce significant concentrations of hematopoietic growth factors in culture and augment hematopoietic colony formation when co-cultured with UCB mononuclear cells. Mice undergoing transplantation with limited numbers of human UCB cells or CD34(+) selected cells demonstrated augmented engraftment when UC-MSCs were co-transplanted. We also explored whether UC-MSCs could be further manipulated by transfection with plasmid-based vectors. Electroporation was used to introduce cDNA and mRNA constructs for GFP into the UC-MSCs. Transfection efficiency was 31% for cDNA and 90% for mRNA. These data show that UC-MSCs represent a reliable, easily accessible, noncontroversial source of MSCs. They can be prepared and cryopreserved under good manufacturing practices (GMP) conditions and are able to enhance human hematopoietic engraftment in SCID mice. Considering their cytokine production and their ability to be easily transfected with plasmid-based vectors, these cells should have broad applicability in human cell-based therapies.

Multiple natural killer (NK) cell-based anticancer therapies are currently under development. Here, we compare the efficiency of genetically modified NK-92 cells expressing chimeric antigen receptors (CARs) at killing NK cell-resistant B-lymphoid leukemia cells to the antibody-dependent cell-mediated cytotoxicity (ADCC) of NK-92 cells expressing a high affinity variant of the IgG Fc receptor (FcγRIII). First, we compared in vitro the abilities of NK-92 cells expressing CD20-targeting CARs to kill primary chronic lymphocytic leukemia (CLL) cells derived from 9 patients with active, untreated disease to the cytotoxicity of NK-92 cells expressing FcγRIII combined with either of the anti-CD20 monoclonal antibodies (mAbs) rituximab or ofatumumab. We found that CAR-expressing NK-92 cells effectively kill NK cell-resistant primary CLL cells and that such a cytotoxic response is significantly stronger than that resulting from ADCC. For studying CAR-expressing NK cell-based immunotherapy in vivo, we established xenograft mouse models of residual leukemia using the human BCR-ABL1+ cell lines SUP-B15 (CD19+CD20−) and TMD-5 (CD19+CD20+), two acute lymphoblastic leukemia (ALL) lines that are resistant to parental NK-92 cells. Intravenous injection of NK-92 cells expressing CD19-targeting CARs eliminated SUP-B15 cells, whereas they had no such effect on TMD-5 cells. However, the intrafemoral injection of NK-92 cells expressing CD19-targeting CAR resulted in the depletion of TMD-5 cells from the bone marrow environment. Comparative studies in which NK-92 cells expressing either CD19- or CD20-targeting CARs were directly injected into subcutaneous CD19+CD20+ Daudi lymphoma xenografts revealed that CD20-targeting CAR is superior to its CD19-specific counterpart in controlling local tumor growth. In summary, we show here that CAR-expressing NK-92 cells can be functionally superior to ADCC (as mediated by anti-CD20 mAbs) in the elimination of primary CLL cells. Moreover, we provide data demonstrating that the systemic administration of CAR-expressing NK-92 cells can control lymphoblastic leukemia in immunocompromised mice. Our results also suggest that the direct injection of CAR-expressing NK-92 cells to neoplastic lesions could be an effective treatment modality against lymphoma.

An emerging treatment option for chronic lymphocytic leukemia (CLL) is to make cytotoxic immune cells express a chimeric antigen receptor (CAR) that recognizes specific surface molecules on CLL cells. Here an mRNA coding for an anti-CD19 CAR was transfected into the NK-92 cell line by electroporation. In contrast to cDNA, mRNA resulted in high transfection efficiency (47.2 ± 8% versus <5% for cDNA) with minimal effect on cell viability. NK-92 cells expressing anti-CD19 CAR killed previously resistant CD19 + BALL cell lines, as well as primary CLL cells and therefore may present a safe, cell-based, targeted treatment for patients with CLL.

Natural killer (NK) cells can be engineered to kill resistant B-lymphoid cell lines and primary B-cell chronic lymphocytic leukemia (B-CLL) cells after transfection with chimeric antigen receptors (CARs) recognizing CD19 or CD20. Here we compared mRNA electroporation with lentiviral vector (LV) transduction for both CARs. Transfection efficiency and cytotoxicity of previously NK-92 resistant CLL cells were significantly higher after mRNA electroporation than after LV transduction. Further cell sorting of LV-transduced NK-92 cells resulted in a highly enriched population of transduced cells with significant target cell lysis. Compared to NK-92 cells, peripheral blood and cord blood cells consistently showed < 10% transfection efficiency with mRNA, while LV transduction varied between 8 and 16% for peripheral blood and 12 and 73% for cord blood. These results suggest that LV should be used to achieve sufficient transgene expression if blood NK cells are considered for CAR transduction. Transfection with mRNA results in clinically relevant levels of transfection only in NK-92 cells.

Natural killer (NK) cell-mediated cytotoxicity can control leukemia relapse while protecting patients from graft-versus-host disease (GVHD) after allogeneic stem cell transplant. Cord blood (CB) is rich in NK cell progenitors with similar properties of proliferation and cytotoxicity as adult blood NK cells. Hence, it is attractive to expand and potentially utilize these cells for adoptive immunotherapy. In this study, CB mononuclear cells were CD3-depleted by immunomagnetic microbead selection to remove T cells. This CD3(dep) CB-MNC fraction was then plated for ex vivo expansion, with or without a feeder layer of irradiated umbilical cord mesenchymal stem cells (UC-MSC), with or without cytokines that have been shown to be critical for NK expansion: IL-2, IL-15, IL-3, and FLT-3L. At an average of 2 weeks of culture, there was significantly higher expansion (64.7 +/- 8.4-fold) of CD56(+)/CD3(-) NK cells in the presence of the UC-MSC feeder layer and cytokines compared to controls (no increase with feeder layer only and 6.4 +/- 1.5-fold increase with cytokines only, P < .05). Contact between CD3(dep) CB-MNC cells and UC-MSC augmented NK expansion. The combination of all 4 cytokines was superior to IL-2 alone or 2 cytokines combinations: mean 64.7 +/- 8.4-fold expansion with 4 cytokines combination versus IL-2 alone, IL-2 + FLT-3L, IL-2 + IL-15 or IL-2 + IL-3 (12.2 +/- 2.0, 14.4 +/- 2.4, 10.4 +/- 4.1, 25.2 +/- 8.1 respectively). We also observed that only fresh CD3(dep) CB-MNC preparations could be expanded reliably, whereas frozen and thawed CD3(dep) CB-MNC cells did not expand consistently (mean fold increase 6.5 +/- 3.2). Cytotoxicity of expanded NK cells was compared with NK cells from fresh and overnight IL-2 activated CD3(dep) CB-MNC. Whereas fresh cells displayed no discernible killing, strong cytotoxicity against K562, Raji, REH, and SUP-B15 cells lines was noted after overnight activation in IL-2. Cytotoxicity of expanded NK cells against Raji, REH, and SUP-B15 was lower, which, however, correlated with a predominant expansion of CD56(+)/CD16(-) cells known to have less cytolytic activity than CD56(+)/CD16(+). To test the transfection efficiency in NK cells, fresh or expanded CD3(dep) CB-MNC cells were electroporated with either DNA or mRNA constructs for GFP. DNA had a low transfection efficiency (<10%), whereas the one for mRNA reached 52%, but at the cost of significant cell death. Our results suggest that CB NK cell progenitors can be expanded to obtain large numbers by using an irradiated feeder of UC-MSC. They maintain an elevated cytotoxic profile, and may be genetically manipulated-all characteristics that make them suitable for cellular therapies.

The Ras protein activates at least three different pathways during early development. Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein. From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral. Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton. To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB. We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton. In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP. We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.