Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry

<div><p>Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The ⁵¹Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.</p></div

Percent specific lysis of K562 cell line using protocol optimized for direct image cytometry assay.

<p>(a) Cytotoxicity against K562 was repeated by image cytometry with reduced target cell number (50,000 cells/well), in a final media volume of 100 μl. After the standard 4-hour incubation the cells were directly resuspended in the 100 μl volume and read using image cytometer. (b) The data shows that with reduced target cell density (Low) the cytotoxicity of NK cells against K562 was significantly higher compared to previous assay with higher target cell density (High) of 100,000 cells/well. The statistical analysis was performed using Wilcoxon matched-pairs signed rank, non-parametric, two-tailed t test and <i>p</i> value of <0.05 was considered significant. The data is plotted as median with range.</p

Natural Killer Cells Stimulated With Pm21 Particles Expand And Biodistribute In Vivo: Clinical Implications For Cancer Treatment

Background aims: Natural killer (NK) cell immunotherapy for treatment of cancer is promising, but requires methods that expand cytotoxic NK cells that persist in circulation and home to disease site. Methods: We developed a particle-based method that is simple, effective and specifically expands cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) both ex vivo and in vivo. This method uses particles prepared from plasma membranes of K562-mb21-41BBL cells, expressing 41BBL and membrane bound interleukin-21 (PM21 particles). Results: Ex vivo, PM21 particles caused specific NK-cell expansion from PBMCs from healthy donors (mean 825-fold, range 163-2216, n = 13 in 14 days) and acute myeloid leukemia patients. The PM21 particles also stimulated in vivo NK cell expansion in NSG mice. Ex vivo pre-activation of PBMCs with PM21 particles (PM21-PBMC) before intraperitoneal (i.p.) injection resulted in 66-fold higher amounts of hNK cells in peripheral blood (PB) of mice compared with unactivated PBMCs on day 12 after injection. In vivo administration of PM21 particles resulted in a dose-dependent increase of PB hNK cells in mice injected i.p. with 2.0 × 106 PM21-PBMCs (11% NK cells). Optimal dose of 800 μg/injection of PM21 particles (twice weekly) with low-dose interleukin 2 (1000 U/thrice weekly) resulted in 470 ± 40 hNK/μL and 95 ± 2% of total hCD45+ cells by day 12 in PB. Furthermore, hNK cells were found in marrow, spleen, lung, liver and brain (day 16 after i.p. PM21/PBMC injection), and mice injected with PM21 particles had higher amounts. Conclusions: The extent of NK cells observed in PB, their persistence and the biodistribution would be relevant for cancer treatment

Determination of target cell lysis by image cytometry.

<p>The figure shows images from the image cytometer and analysis of fluorescence data using FCS express, to derive live target cell counts. The number of live target cells in the culture was determined by plotting fluorescence intensity of target cells from each E:T ratio compared to spontaneous control. The live K562 cells are shown in blue circles and the lysed cells and apoptotic bodies are highlighted in red circles in the images. Representative fluorescence histograms used for deriving live target cell counts are shown for each tumor cell lines (K562, 721.221 and Jurkat cells).</p

Comparison of percent specific lysis obtained by calcein release assay and image cytometry.

<p>Standard calcein release assay and image cytometry were performed within the same assay to determine NK cell killing of K562, 721.221 and Jurkat. The data is shown for each tumor cell line using NK cells expanded from 5 donors (n = 5). Cytotoxicity assay was performed in triplicate for each NK cell donor. Each of the replicate is presented in the plots. The statistical analysis was performed using Wilcoxon matched-pairs signed rank, non-parametric, two-tailed t test and <i>p</i> value of <0.05 was considered significant. The data is plotted as median with range.</p

Sensitivity of calcein release assay.

<p>(a) Dynamic range for the calcein release assay was determined for neuroblastoma and leukemia tumor targets (K562, SK-N-BE(2), CHP134, CHLA155, IMR32, Jurkat, 721.221 and Nalm 6). The cell lines were stained with Calcein AM, and seeded in 96 well plates to generate maximum release (lysis with 1% TritonX-100) and spontaneous release data. After 4 hours the supernatant fluorescence was measured. The maximum release was normalized to 100% and the spontaneous release is represented as % of maximum. (b) Illustration of calcein release from target cells upon lysis by NK cells following necrosis-like and apoptotic death. (c) Bright-field and fluorescence overlay images of calcein release from CHP 134 cells undergoing necrosis-like death following interaction with NK cells. (d) Bright-field and fluorescent overlay images of calcein release from some K562 cells undergoing apoptotic death following interaction with NK cells. The images were derived from live Nikon Biostation IQ-M videos.</p