Laurent Boissel

Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells

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+ B-ALL cell lines, as well as primary CLL cells and therefore may present a safe, cell-based, targeted treatment for patients with CLL.

Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells

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.

Umbilical Cord Mesenchymal Stem Cells Increase Expansion of Cord Blood Natural Killer 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.

Comparison of mRNA and lentiviral based transfection of natural killer cells with chimeric antigen receptors recognizing lymphoid antigens

Abstract 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.

Variable contribution of monoclonal antibodies to ADCC in patients with chronic lymphocytic leukemia.

Monoclonal antibodies (mAbs) are increasingly used in treatment protocols for chronic lymphocytic leukemia (CLL). Here we determined (i) the extent of antibody-dependent cellular cytotoxicity (ADCC) of four different mAbs against primary CLL cells, (ii) whether ADCC correlates with antigen density on CLL cells, and (iii) whether allogeneic natural killer (NK) cells display superior ADCC than autologous. Effector cells for ADCC were (i) NK-92 cells not expressing FcR, (ii) NK-92 cells transfected with a high-affinity Fc receptor, (iii) autologous NK cells from patients with CLL, (iv) allogeneic NK cells. Results suggest that ADCC contributes to killing of CLL cells by anti-CD20 antibodies (rituximab and veltuzumab), whereas mAbs against CD22 (epratuzumab) and CD23 (lumiliximab) showed minimal ADCC. The magnitude of anti-CD20 mediated ADCC did not correlate with antigen density of CD20. ADCC was not influenced by the FcR genotype expressed by autologous NK cells. Allogeneic NK cells were superior to autologous NK cells in killing primary CLL cells.

An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele

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.

Abstract 2302: An ‘off the shelf,’ GMP-grade, IL-2-independent NK cell line expressing the high-affinity Fc-receptor to augment antibody therapeutics

A number of approved IgG1 monoclonal antibodies (mAb) used in cancer therapy, including trastuzumab, cetuximab, and rituximab, depend on natural killer (NK) cell-mediated antibody dependent cell-mediated cytotoxicity (ADCC) to induce cancer cell death, mediated through the Fcγ receptor (FcγR). Multiple studies report that survival benefit is significantly improved in cancer patients receiving IgG1 antibody therapy who have NK cells that express the high-affinity FcγRIIIa receptor (CD16-158V). Only 12% of the normal human population, however, is homozygous for expression of this high-affinity variant CD16. We hypothesized that infusion of an NK-92 cell line modified to express CD16-158V (high-affinity NK cells [haNK]) in combination with an IgG1 mAb may result in superior antitumor effects compared with IgG1 mAb alone. A GMP-grade plasmid-transfected variant of NK-92 expressing the high-affinity CD16 receptor was developed utilizing a novel transfection vector containing the erIL-2 gene, enabling the resulting haNK cells to grow independently of IL-2. Using limiting dilution, clones having identical surface molecule expression to the parent cell line were generated and shown to have long-term expression of CD16. The haNK cells displayed high ADCC in combination with rituximab, trastuzumab, and daratumumab against target cell lines that were not killed by the parental NK-92 cells. Of note, despite non-viral transfection, the plasmid is fully integrated into chromosome 5. The haNK cell line secretes about 200 pg/106 cells of IL-2, and like the parental NK-92 cell line, produces enough IFNγ to potentially stimulate an adaptive immune response. A GMP-grade master cell bank of haNK cells has been established; haNK cells can be cryopreserved with retention of ADCC activity. In vivo studies demonstrated the efficacy of haNK cells. In a multiple myeloma model of CD38+ NCI-H929 cells xenografted into NOD/SCID gamma null (NSG) mice, treatment with haNK cells plus the anti-CD38 mAb daratumumab led to prolonged survival versus mice treated with haNK cells plus an isotype control (log rank p = 0.0169), demonstrating therapeutically relevant in vivo ADCC. haNK cells have also been transfected with various CARs using mRNA, thereby enabling this platform to make a bispecific effector cell product. In summary, haNK cells are a potent and versatile platform for cellular immunotherapy, and clinical trials of these novel “off the shelf” high-affinity NK cells are scheduled for 2016. Citation Format: Laurent Boissel, Hans Klingemann, Kerry Campbell, Karen Nichols, Frances Toneguzzo, Paula Marcus, Brent Williams, Armand Keating, Patrick Soon-Shiong. An ‘off the shelf,’ GMP-grade, IL-2-independent NK cell line expressing the high-affinity Fc-receptor to augment antibody therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2302.

Abstract 2046: EZH2 inhibition targets a stem cell population in a canine urothelial carcinoma model

Recent studies have revealed that aberrant epigenetic events contribute to the development and maintenance of a malignant phenotype in urothelial carcinoma (UC) of the bladder, and many other tumor types. Thus, drugs capable of reversing epigenetic alterations have been brought to focus, and in the clinic epigenetic therapies demonstrate efficacy in the treatment of particular hematopoietic tumors. However, despite encouraging results from mouse xenograft studies, the clinical application of these agents to the treatment of solid tumors has been less successful. It is clear that new models are needed for the development of therapeutic strategies that target the solid tumor epigenome. Canine UC that develops commonly and spontaneously in dogs may provide a relevant model to directly inform new clinical protocols, to permit discovery and testing of clinical biomarkers, and, ultimately, to predict the response of human tumors in the clinic. Although human and canine UC share histopathologic characteristics, molecular features, and response to medical therapy, little is known about the canine UC epigenetic status. In human UC, EZH2 is overexpressed and, as a consequence of its enzymatic activity that involves the tri-methylation of histone H3 (H3K27me3), is responsible for silencing a large number of genes, including known tumor suppressors. We have determined that EZH2 is also overexpressed in canine UC. Therefore, we hypothesize that EZH2 provides a therapeutic target for UC. Pharmacological inhibition of EZH2 (DZNep) as well as targeted knockdown inhibits cell growth and induces apoptosis in canine UC cells in vitro in anchorage-dependent (2D) and anchorage-independent (3D) cultures. By conducting these experiments in parallel with human UC cell lines, we showed that sensitivity to inhibitor is comparable between human and canine UC cell lines. Upon DZNep treatment, EZH2 is reduced and its activity is inhibited, as evidenced by both decreased global H3K27me3 and upregulation of a set of genes that are commonly silenced by EZH2. In parallel, we showed that inhibition of EZH2 reduced the sphere forming capacity of canine UC cells, suggesting an inhibitory effect in the growth of the stem cell subpopulation. Taking into account that this population is responsible for dissemination, metastasis and emergence of drug resistance, these results support the idea that EZH2 targeting in UC could be a beneficial addition to the existing therapeutic modalities. According to our data, the molecular underpinnings of spontaneous UC are also shared between dogs and humans. Therefore canine UC provides a means to understand epigenetic dysregulation in the urothelium as well as to investigate its clinical targeting. This provides the opportunity to easily evaluate tumor response in vivo and obtain serial tissue samples during treatment. Thus canine studies can provide data that are not easily obtained in human trials and may inform clinical development. Citation Format: Parthena Foltopoulou, Monica Betancur-Boissel, Laurent Boissel, Manar A. AbdelMageed, Philip N. Tsichlis, Elizabeth A. McNiel. EZH2 inhibition targets a stem cell population in a canine urothelial carcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2046. doi:10.1158/1538-7445.AM2014-2046