Gene-modified Natural Killer Cells Research Articles

Non-Viral Sleeping Beauty Transposon Engineered CD19-CAR-NK Cells Show a Safe Genomic Integration Profile and High Antileukemic Efficiency

Background: Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity. Recently, we and others showed that virally transduced NK cells equipped with a synthetic chimeric antigen receptor (CAR) targeting CD19 induced enhanced killing of acute lymphoblastic leukemia (ALL) cells. Here, we demonstrate for the first time that primary NK cells can be engineered using the non-viral Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-CAR with a safe genomic integration profile and high anti-leukemic efficiency in vitro and in vivo.Methods: Primary NK cells were isolated from PBMCs from healthy donors. SB transposons vectorized as minicircles (MC), which encode either a Venus fluorescent protein or a CD19-CAR together with truncated EGFR (tEGFR) as a marker, were introduced in combination with the hyperactive SB100X transposase into primary NK cells via nucleofection. The genetically engineered NK cells were expanded using IL-15 cytokine stimulation under feeder-cell free conditions. Vector integration sites were mapped by analyzing the genomic region around each insertion site in genomic DNA from long-term cultivated gene-modified NK cells, engineered ether by lentiviral (LV) or SB-based technology. Stable gene delivery and biological activity were monitored by flow cytometry and cytotoxicity of CD19-CAR NK cells against CD19-positive ALL and CD19-negative cell lines.Results:Applying a protocol optimized with respect to nucleofection pulses, time points and plasmid ratios, primary NK cells showed long-lasting Venus expression (up to 50%) upon SB-mediated gene delivery with similar viability as non-treated (NT) NK cells during feeder-cell free ex-vivo expansion using IL-15. Likewise, SB transposon-engineered CD19-CAR NK cells displayed high viability, durable transgene expression (Fig 1 A), and no significant change in the NK cell phenotype profile.Next, we assessed vector integration into genomic safe harbors (GSH). GSH are defined as regions of human chromosomes that fulfill the following five criteria: not ultraconserved, >300 kb away from miRNA genes, >50 kb away from transcriptional start sites (TSS), >300 kb away from genes involved in cancer and outside transcription units. CD19-CAR NK cells generated using SB100X showed a significantly higher frequency of vector integration into GSH compared to LV-transduced CAR-NK cells and a significantly more-close to random nucleotide frequency (computer-generated random positions in the genome map to GSHs; random 43.68%; LV 14.78%, SB100X 23.99%; p<0.05) (Fig 1 B).MC.CD19-CAR NK cells generated with the SB platform demonstrated significantly higher cytotoxicity compared to NT NK cells against CD19-positive Sup-B15 ALL cells after long-term cultivation for two to three weeks and no loss of natural intrinsic cytotoxicity. After 4-hour co-culture, significantly enhanced specific tumor cell lysis was found for MC.CD19-CAR NK cells vs NT NK cells at all effector to target cell ratios (E:T) tested (E:T 20:1 83.88% vs 43.13%; E:T 10:1 75.18% vs 31.32%; E:T 5:1 67.38 vs 32.22%; E:T 1:1 42.54 vs 10.19%; p<0.05) (Fig 1 C). With regard to intrinsic natural cytotoxicity of NK cells, no significant decrease in cell killing was overserved for SB-gene-modified CD19-CAR NK cells compared to NT NK cells against CD19-negative K562 cells (E:T 5:1 83%; p<0.05) (Fig 1 D).Significantly enhanced antitumor potential of SB-generated CD19-CAR NK cells was confirmed in a systemic CD19-positive lymphoma xenograft model (NSG-Nalm-6/Luc) in vivo. After injection of 0.5x10 6 tumor cells per mouse and lymphoma engraftment, animals were treated with a single dose of 10x10 6 SB-modified CD19-CAR NK cells pooled from three different donors with a mean tEGFR/CAR expression of 34%. MC.CD19-CAR NK cell therapy resulted in rapid lymphoma eradication in all treated mice (n=4; p<0.05), whereas mice receiving similar amounts of NT NK cells showed progressive lymphoma growth comparable to untreated control mice (Fig 1 E-F).Conclusion: Taken together, the Sleeping Beauty transposon system represents an innovative gene therapy approach for non-viral engineering of safe, highly functional and relatively cost-efficient CAR-NK cells that may not only be suitable for ALL therapy but also for a broad range of other applications in cancer therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Engineering the TGFβ Receptor to Enhance the Therapeutic Potential of Natural Killer Cells as an Immunotherapy for Neuroblastoma.

The ability of natural killer (NK) cells to lyse allogeneic targets, without the need for explicit matching or priming, makes them an attractive platform for cell-based immunotherapy. Umbilical cord blood is a practical source for generating banks of such third-party NK cells for "off-the-shelf" cell therapy applications. NK cells are highly cytolytic, and their potent antitumor effects can be rapidly triggered by a lack of HLA expression on interacting target cells, as is the case for a majority of solid tumors, including neuroblastoma. Neuroblastoma is a leading cause of pediatric cancer-related deaths and an ideal candidate for NK-cell therapy. However, the antitumor efficacy of NK cells is limited by immunosuppressive cytokines in the tumor microenvironment, such as TGFβ, which impair NK cell function and survival. To overcome this, we genetically modified NK cells to express variant TGFβ receptors, which couple a mutant TGFβ dominant-negative receptor to NK-specific activating domains. We hypothesized that with these engineered receptors, inhibitory TGFβ signals are effectively converted to activating signals. Modified NK cells exhibited higher cytotoxic activity against neuroblastoma in a TGFβ-rich environment in vitro and superior progression-free survival in vivo, as compared with their unmodified controls. Our results support the development of "off-the-shelf" gene-modified NK cells, that overcome TGFβ-mediated immune evasion, in patients with neuroblastoma and other TGFβ-secreting malignancies.

Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G

ABSTRACTActivated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2 in activated NK cells increased their responses to GD2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro.We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

Genetically Engineered Natural Killer Cells as a Means for Adoptive Tumor Immunotherapy.

Natural killer (NK) cells are lymphoid cells of the innate immune system; they stand at the first defense line against viruses and transformed cells. NK cells use an array of germline-encoded activating and inhibitory receptors that sense virus-infected cells or malignant cells displaying altered surface expression of activating and inhibitory NK cell ligands. They exert potent cytotoxic responses to cellular targets and thus are candidate effector cells for immunotherapy of cancer. In particular, the genetic engineering of NK cells with chimeric antigen receptors (CARs) against surface-expressed tumor-associated antigens (TAAs) seems promising. In the allogeneic context, gene-modified NK cells compared to T cells may be superior because they are short-lived effector cells and do not cause graft-versus-host disease. Furthermore, their anti-tumoral activity can be augmented by combinatorial use with therapeutic antibodies, chemotherapeutics, and radiation. Today, efforts are being undertaken for large-scale NK-cell expansion and their genetic engineering for adoptive cell transfer. With the recent advances in understanding the complex biological interactions that regulate NK cells, it is expected that the genetic engineering of NK cells and a combinatorial blockade of immune evasion mechanisms are required to exploit the full potential of NK-cell-based immunotherapies.

Gene Modification of Human Natural Killer Cells Using a Retroviral Vector.

As part of the innate immune system, natural killer (NK) cells are regarded as promising effector cells for adoptive cell therapy approaches to treat patients with cancer. In some cases, genetic modification of the NK cells may be considered but such manipulation has to be integrated into the expansion method to allow the generation of clinically relevant numbers of gene-modified NK cells. Therefore, an efficient gene transfer procedure is needed.Our group developed a retrovirus-based transduction protocol capable of robust expansion of gene-modified NK cells with a high rate of transgene expression. Actively dividing cells is a prerequisite for efficient gene transfer when using a retroviral vector. In the procedure presented here, strong activation of the NK cells was provided by a combination of IL-15 and the K-562 feeder cells. Beside the interest in developing a simple procedure compliant with good manufacturing practice (GMP) for the production of therapeutic products, this approach also provides a valuable means of generating genetically modified primary NK cells for future preclinical studies.

Development of Optimized Protocol for Generation of NK Cells Expressing Chimeric Antigen Receptors from Hematopoietic Stem Cells for Cancer Immunotherapy

Background : Natural Killer (NK) cells are innate immune cells that mediate cytotoxicity against tumor and virus-infected cells, and represent a very promising source for adoptive cellular approaches for cancer immunotherapy. Extensive research has been conducted, including clinical trials, attempting to harness their properties. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer in mature NK cells is very limited. We have previously published a protocol for generation of human NK cells from gene-modified hematopoietic stem cells (HSC) isolated from umbilical cord blood. Generation of NK cells from HSC provides the opportunity of generation of younger NK cells and expansion of specific gene-modified clones starting from a smaller number of previously isolated and cryopreserved initial cells, with added advantage of generation of multiple batches from the same donor. Chimeric antigen receptors (CAR) are engineered fusion proteins that combine the antigen specificity of antigen-binding moieties of monoclonal antibodies and intracellular activation motifs capable to activate immune cells. Preliminary evidence suggests that NK cells with specificity directed by second-generation CAR may have enhanced cytotoxicity. The goal is to develop a protocol with maximal generation of CAR-expressing NK cells from human HSC for clinical applications. We have evaluated the use of HSC isolated from G-CSF-mobilized apheresed peripheral blood mononuclear cells (PBSC), the elimination of serum in culture media and the elimination of feeder stroma.Significance : Development of a protocol for clinical translation and large-scale good manufacturing practice (GMP) compatibility, maximally generating functional CAR-expressing NK cells. G-CSF mobilized peripheral blood stem cells (PBSC) were used because of availability of larger HSC numbers, increasing safety and efficacy. Changes in culture media based on available literature were evaluated to promote generation of larger number of cells. Human AB serum and serum free media were tested to determine the cell yield for large-scale GMP-compatible protocol.Methods : A third-generation lentiviral vector co-delivering CD19-specific CAR and enhanced green fluorescent protein (EGFP) was used for gene modification of primary human PBSC. Gene-modified cells were then co-cultured with OP9-DL1 stromal cells over 35-40 days in six different culture media conditions for evaluation. Medium "A" was our previously published protocol and consisted of alpha-MEM enriched with 20% of fetal bovine serum and recombinant human cytokines SCF 5ng/mL, Flt3L 5ng/mL, IL-7 5ng/mL, and IL-15 10ng/mL. Medium "B" was AIM V enriched with 10% of human AB serum and cytokines SCF 5ng/mL, Flt3L 5ng/mL, IL-7 20ng/mL, and IL-15 50ng/mL. Medium "C" was similar to medium "B" excluding human AB serum. After 10 days of culture, IL-2 10ng/mL was added to all three media ("plus") creating six different conditions. Flow cytometry was used for detection of EGFP expression and NK cell surface markers. Digital droplet PCR was used for analysis of number of integrated viral copies. Feeder-free culture conditions were developed with the addition of recombinant human IGF-1 100ng/mL to AIM V culture media enriched with SCF, Flt-3 and IL-15.Results : NK cell differentiation was achieved in all conditions. Feeder-free conditions seemed to present mature NK cells earlier (days 25-30) than stromal co-culture (days 35-45), but lower cell yield. As previously reported, PBSC had lower yields of NK differentiation as compared to umbilical cord blood, but higher concentrations of IL-7 and IL-15 rescued the differentiation. Total cell yields were 100-220-fold expansions, with highest counts recovered from conditions with higher IL-7 and IL-15. The removal of serum and addition of IL-2 did not seem to affect differentiation or proliferation. CD56+/CD16+/CD94+ NK cells were present in 10-40% of all CD56+ cells.Conclusions : Large-scale GMP-compatible generation of clinically-relevant numbers of gene-modified NK cells from HSC is feasible. Higher doses of cytokines IL-7 and IL-15 successfully increase the yield of NK cells from PBSC. Absence of serum did not decrease differentiation or proliferation. PBSC showed folds of expansion and NK cell differentiation comparable to those obtained from umbilical cord blood. DisclosuresNo relevant conflicts of interest to declare.

Human Natural Killer Cells Exhibit Negative Regulatory Function by Ectopic Expression of hFoxp3 Gene

Foxp3 is a key marker of CD4CD25 regulatory T cells and appears highly specific for regulatory T cells. Human dendritic cells transfected with foxp3 gene also exhibit immunosuppressive functions. We want to understand whether natural killer (NK) cells could be endowed with regulatory properties by transduction of Foxp3 gene. A recombinant vector (pRV.GFP Foxp3) or control vector (pRV.GFP WWRR) was transferred into NKL/NK-92 cells by an electroporation method. The hFoxp3 gene-modified NK cells were characterized with regard to their proliferation, cytokine production, and cytotoxicity and their regulatory effects on activated human peripheral blood mononuclear cells (hPBMCs) in vitro and trans vivo delayed-type hypersensitivity assay. We found that the ectopic expression of hFoxp3 in human NK cells resulted in the high production of the immunosuppressive cytokine, interleukin (IL)-10. Luciferase reporter assay showed that the expression of IL-10 is directly regulated by Foxp3. We observed that NKL.Foxp3 cells inhibited the proliferation and activation of phorbol-12-myristate-13-acetate/ionomycin-stimulated hPBMCs; furthermore, NKL.Foxp3 cells significantly suppressed the delayed-type hypersensitivity response, which was induced by anti-CD3 monoclonal antibody-activated hPBMCs. NKL.Foxp3 cell-mediated negative regulatory function was dependent on IL-10 production. Our findings indicated that NK cells acquired IL-10 phenotype by transduction with foxp3 gene and provided evidence that Foxp3 could exert regulatory function not only in regulatory T cells but also in NK cells. These results suggested that Foxp3 gene-modified NK cells might be potential usefulness on graft-versus-host disease or some autoimmune diseases.

NK cells engineered to express a GD2‐specific antigen receptor display built‐in ADCC‐like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD2, which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD2-specific chimeric antigen receptor (CAR) comprising an anti-GD2 ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD2 expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD2-specific antibody or anti-idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2-specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.