ALBERT

Description: Introduction: We are studying NK cell immunotherapy to treat acute myeloid leukemia (AML) and have focused on NK-92 and KHYG-1, CD16(-) human malignant NK cell lines. Phase I NK-92 trials show minimal toxicity; KHYG-1 has not been tested in humans. Here, we investigated modulation of cytotoxicity of NK cell lines against primary AML blasts and cell lines with monoclonal antibodies (mAb) directed against natural cytotoxicity receptors. Methods: NK cytotoxicity was assessed with a standard 4 hour Cr51 release assay at an effector to target (E:T) ratio of 10:1. NK lines were incubated with and without isotype control and mAbs against NKp30, NKp44 at various doses (0.001-10 µg/ml) for 1 hour and washed with medium prior to cytotoxicity assays. The student’s t-test was used to compare cytotoxicity data. Target cells were incubated with 100 µCi Cr51 and cell supernatants assayed on a gamma counter. NK targets (leukemic and esophageal cancer) were evaluated for Fcγ receptor expression by flow cytometry. To test the cytotoxic effect on in vivo proliferation, OCI/AML5 cells were co-incubated with irradiated KHYG-1 (iKHYG-1) +/-1 µg/ml NKp30 pretreatment for 4 hours at a 10:1 E:T ratio and injected ip into NOD/SCID gamma null (NSG) mice with survival as an endpoint analyzed with the log rank test. Results: NK-92 and KHYG-1 were both highly cytotoxic against K562 with moderate killing of OCI/AML3 and KG1 and KG1a. OCI/AML5 was highly sensitive to killing by NK-92, but resistant to KHYG-1. Pretreatment of NK-92 with mAbs against NKp30, NKp44 (10 µg/ml) yielded small increases in cytotoxicity against leukemic cell lines with NKp30 pretreatment only. Pretreatment of KHYG-1 with 10 µg/ml of anti-NKp30 or anti-NKp44 mediated fold increases in cytotoxicity above isotype control against 4 leukemia cell line targets and 4 primary AML samples (Table1). Anti-NKp30 and anti-NKp44 pretreatment of NK-92 and KHYG-1 did not enhance killing of a panel of esophageal cancer cell lines. Immunophenotyping cancer cell lines showed high expression of Fcγ receptor II (CD32), but very low expression of Fcγ receptor I (CD64) or III (CD16) on leukemia lines (K562, OCI/AML3, OCI/AML5, KG1 and KG1a), and no expression of Fcγ receptors on esophageal lines (OE-33, FLO-1, KYAE-1, SKGT-4). Regression analysis of the relationship between cytotoxic enhancement and CD32 expression of targets revealed a strong correlation for NKp30 (p

Description: Abstract 4300 Introduction: Outcome of patients with acute myeloid leukemia (AML) remains poor because many relapse from residual leukemic stem cells (LSCs) enriched in the CD34+CD38- fraction of AML blasts. NK-92 is a human permanent natural killer (NK) cell line in phase I clinical trials for relapsed and refractory malignancies. We recently showed that NK-92 targets LSCs preferentially over bulk leukemia in the cell line KG1 (Cytotherapy. 2010 Mar 15). Here, we evaluate the action of NK-92 and another NK cell line, KHYG-1, against five AML cell lines and five primary AML samples by the chromium release and methylcellulose cytotoxicity assays to determine the mechanism of recognition and killing and the effect on leukemic stem cells. Results: Using a 4 hour chromium release assay (CRA), the highest effector:target (E:T) ratio tested (25:1) for NK-92 and KHYG-1 against cell line targets revealed % lysis as follows: K562: 81.2+/−5.4%; 82.2 +/−3.9%; KG1: 41.0+/−7.2%; 37.3% +/−3.6; OCI/AML2: 37.3+/−22.7%; 33.0+/−13.9%; OCI/AML3: 33.8+/−4.5%; 51.0+/− 6.8% and OCI/AML5: 99.0+/−4.9%; 52.9 +/− 5.6%, respectively. Killing by NK-92 and KHYG-1 was completely inhibited by calcium chelation using 4 mM EGTA for all cell lines tested. Blockade of class I HLA on target cells using 10 ug/ml of anti-class I monoclonal antibody did not affect killing by NK-92 and KHYG-1 except for a decrease in killing of OCI/AML5 by both NK-92 and KHYG-1 from 99.0 +/− 4.9% to 39.0 +/−3.2% and 52.9+/−5.6% to 19.6 +/− 6.25%, respectively. Blockade of NKG2D using 10 ug/ml of anti-NKG2D monoclonal antibody did not significantly affect killing of AML cell lines by NK-92 or KHYG-1. Five primary AML blast samples treated with NK-92 at 25:1 E:T yielded only slight to moderate degrees of cytotoxicity by CRA: 15.6 +/−12.7%, 42.3+/−3.6%, 29.8 +/−3.6%, 43.9 +/− 1.47%, 42.6 +/− 0.1%lysis. KHYG-1 at 25:1 E:T had minimal killing of this panel of AML blasts by CRA: 1.27 +/−21.9%, 9.8+/−2.2%, 5.2+/−2.5%, 17.1+/−2.8%, 8.5+/−3.3% lysis. Blockade of class I HLA did not affect killing of primary AML blasts by NK-92, but the fourth sample only was rendered more sensitive to killing by KHYG-1 from 17.1 +/−2.8% to 35.8 +/−1.2% lysis. Blockade of NKG2D with 10 ug/ml anti-NKG2D monoclonal antibody did not significantly affect killing of primary AML samples by NK-92 or KHYG-1. To further assess killing by NK-92 of LSCs from primary AML, we used a methylcellulose cytotoxicity assay (MCA) established previously by our lab. The MCA showed that NK-92 at 25:1 E:T eliminated clonogenic growth of 3/5 primary AML blast samples with minimal colony growth in 2/5 with % cytotoxicity of 100 +/−0%, 86.3 +/− 2.3%, 98.4 +/−2.8%, 100 +/− 0% and 100 +/−0%, demonstrating higher toxicity than obtained with the CRA. Primary AML derived CD34+CD38- sorted LSCs were more sensitive to killing than CD34+CD38+ blasts by NK-92 in a 4 hour CRA at 1:1 E:T: 58.9+/−11.5%, 20.3 +/−1.71%; 5:1: 78.3 +/−9.7%, 43.5+/−11.1% and 10:1: 72.9+/−5.6%, 38.5 +/−2.4% lysis. Summary and conclusion: We demonstrate cytotoxicity of NK-92 and KHYG-1 against a range of AML targets and show greater killing of primary AML blasts by NK-92. The mechanism of cytotoxicity for both cell lines is primarily by granule exocytosis and is NKG2D independent. Unexpectedly, blockade of class I HLA resulted in reduced killing of OCI/AML5 by both NK-92 and KHYG-1, suggesting the presence of an activating KIR receptor common to both cell lines. One of five primary AML samples had increased killing by KHYG-1, suggesting KIR mediated inhibition. Further assessment of NK-92 against LSCs by the MCA demonstrated greater cytotoxicity than with the CRA and indicated preferential killing of LSCs. This finding was confirmed using the CRA with sorted immunophenotypically defined CD34+CD38- LSCs and CD34+CD38+ blasts. Our findings support the use of NK-92 in the treatment of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1854 Introduction: Human NK cell lines NK-92 and KHYG-1 exhibit cytotoxicity against a broad range of tumor types in vitro, including multiple myeloma (MM). To further test efficacy of the NK lines against MM, we developed a bioluminescent mouse model that recapitulates clinical MM using the human U266 MM cell line transduced to express GFP and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow (BM) engraftment. Results: In a pilot study in which 2×106 U266 cells were injected intravenously into NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice, CD138+ MM cells engrafted in BM, with no detectable engraftment in the liver, lungs, spleen, heart, or kidneys by anti-CD138 immunohistochemistry staining at 10 weeks. We used the U266eGFPluc bioluminescent NSG mouse model to evaluate efficacy of NK-92 cell therapy in vivo. We gave 10×106 NK-92 cells every 5 days to a total dose of 50×106 cells 7 days after MM injection. Tumor burden was monitored weekly by bioluminescence imaging 4 weeks after MM inoculation using the IVIS Imaging System, and LivingImage™ Software was used to acquire images and quantify bioluminescence. We showed that U266eGFPluc cells localized to BM and spine, reflecting MM pathophysiology. Disease burden in the NK-92 treated group was consistently lower than controls over time and significantly lower at 8 weeks (Dorsal and Ventral Mann-Whitney p=0.0381) whereas for KHYG-1, the signal increased slightly over control, but was not significant at 8 weeks (Mann-Whitney Dorsal p=0.540 and Ventral p=0.247). Clinical disease progression in MM control mice correlated with IVIS signal intensity at week 11 (r2=0.4; F test p=0.0279). Engraftment was determined by sacrificing mice at 10 weeks and analyzing BM for GFP+ cells by flow cytometry. Engraftment of MM cells in BM was as follows (mean+/− SEM): control (5 +/− 1.9%), NK-92 (0.24 +/− 0.19%) and KHYG-1 (5.2 +/− 1.6%) showing a trend toward a significant decrease in mean engraftment for the NK-92 group versus control (unpaired student's t test p=0.055), but not for KHYG-1 (p=0.939). One of 6 control mice had low engraftment with U266eGFPluc at 10 weeks increasing the variance of the control mean. There was a statistically significant decrease in median engraftment in the NK-92 group (Mann-Whitney p=0.019), but not for KHYG-1 (p =0.792) (Figure). GFP BM engraftment corresponded with bioluminescence detected in R and L BM by IVIS. Presence of NK cells in BM was detected in only 1/3 NK-92 mice tested (0.2%) and in none of the KHYG-1 mice at 10 weeks. To assess biodistribution of KHYG-1 we injected 10×106 CFSE-labeled KHYG-1 via tail vein into healthy NSG mice. Blood and organ samples were collected 8 and 24 hours later and analyzed by flow cytometry. We detected CFSE-labeled KHYG-1 primarily in liver, blood and lung, less in kidney, and none in heart, spleen or BM. Conclusion: We have established a human MM cell line xenograft model in NSG mice comparable to clinical disease. Treatment efficacy can be monitored in live NSG mice by IVIS imaging technology and tumor burden at sacrifice can be determined by GFP detection. MM progression was reduced by NK-92, but not KHYG-1 as measured by bioluminescence and reduction of engrafted U266eGFPluc cells. We have shown that a MM xenograft model can screen for in vivo efficacy of immune therapies for MM. Our results indicate that NK-92 is a potentially effective therapy for MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1909 Introduction: Patients with acute myeloid leukemia (AML) in remission relapse frequently from residual leukemic stem cells (LSCs) that are CD34+CD38-CD123+. NK-92 is an infusible CD16- malignant NK cell line cytotoxic against AML and after irradiation for prevention of NK cell malignancy has minimal adverse events in Phase I trials. Here, we tested irradiated NK-92 (iNK-92) against primary AML in a NOD/SCID gamma null (NSG) xenograft model. To optimize killing of LSCs in vitro we utilized a gene modified CD16+NK-92 against the CD123+ leukemia cell line OCI/AML5 treated with and without an anti-CD123 monoclonal antibody (7G3) to facilitate antibody dependent cell mediated cytotoxicity (ADCC). Methods: NSG mice were irradiated with 225 or 325 cGy before infusion with primary AML blasts (1 or 3×10e6 cells). iNK-92 was started 1 or 10 days post AML infusion and given 1–3 times weekly (15 or 20×10e6 cells/dose) to a total dose of 60–75 ×10e6 cells/mouse. In vitro cytotoxicity was measured by the chromium release assay. OCI/AML5 or primary AML was pretreated with 7G3 or anti-Class I antibody respectively at 10 ug/ml 2 hours before ADCC assays. Results: We developed an NSG AML xenograft model using a CD34+CD38+ primary AML sample containing a small fraction of CD34+CD38- cells that were predominantly CD123+. 3×10e6 primary cells induced leukemia in NSG mice by 6 weeks and maintained comparable potency up to quaternary transplantations with a stable immunophenotype. In vitro cytotoxicity against LSCs was assessed by treating 1×10e6 first passage BM derived primary AML +/− 5×10e6 iNK-92 ×4 hours and injecting into 2 cohorts of 5 mice. At 6 weeks mice were sacrificed and bone marrow harvested. Average leukemic engraftment in the iNK-92 group (79.8, 3.48, 92.1, 81.3, 86.1, Av= 68.6%) was less than untreated AML inoculated group (95.0, 93.4, 19.4, 95, 97.3, Av=80.0%), but not statistically significant (p=0.62). Removing one poorly engrafted outlier mouse from each group yielded a higher engraftment in the control (Av=95.1%) versus the treatment group (Av=84.8%) and was significant (p=0.011). To test iNK-92 to treat engrafted leukemia, NSG mice were infused with 3×10e6 AML cells starting day 10 with 20×10e6 iNK-92 weekly ×6 doses. Survival was improved in the treatment group to near statistical significance (p=0.055), but all mice ultimately succumbed to disease. Addition of IL-2 to iNK-92 did not improve outcomes (p=0.13). 3×10e6 primary AML cells were also infused into 2 cohorts of 4 mice and treated +/− iNK-92 from day 2 and given 15×10e6 cells twice weekly to 75×10e6 total dose. BM (1×10e6 cells) from each of 4 primary recipients in control and treatment was serially transplanted 1:1 into four new NSG mice. BM engraftment occurred in all AML only cohort secondary mice (80.8, 93.3, 80.4, 96.4 Av=87.7%) while one mouse from iNK-92 group was leukemia free with engraftment at background levels of non-injected mice (96.4, 94.7, 1.8, 95.7 Av=72.2%). Proportion of LSCs in secondary transplanted mice was: AML (8.01, 9.48, 8.66, 5.25, Av=7.85%) and AML + iNK-92 (7.13, 3.46, 0.03, 4.0 Av=3.66%) which was statistically significant (p=0.05). CD16+NK-92 killed CD123+ OCI/AML5 cells at effector:target (E:T) ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 35.0 +/−4.0, 9.0+/−6.6, -2.0 +/−0.1, 1.7 +/−3.3) and was significantly enhanced (2–6x) when targets were coated with anti-CD123 mAb (% lysis +/−SD: 64.3 +/−3.1, 48.5 +/−4.1, 20.9 +/−0.1, 10.1+/−3.3). Further, CD16+NK-92 also killed primary AML at E:T ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 34.7 +/−4.6, 15.6 +/− 4.7, 11.5 +/− 2.0, 7.7 +/−1.6) which was significantly enhanced (2–3x) by coating with anti-Class I monoclonal antibodies (% lysis +/−SD: 64.8 +/−10.4, 31.1 +/− 8.9, 30.2 +/− 9.4, 23.9+/−2.8). Conclusion: iNK-92 reduces engraftment of AML and improves survival in a primary AML xenograft model. CD16+NK-92 cytotoxicity against primary AML is enhanced with anti-class I antibodies via ADCC, demonstrating that CD16+NK-92 can be redirected against bulk primary leukemia using a highly expressed cell surface marker. Finally, CD16+NK-92 cytotoxicity can be improved against OCI/AML5 with anti-CD123 antibodies via ADCC, providing the first proof-of-principle for the targeting of leukemic stem cells by combining humoral and cellular approaches. Disclosures: No relevant conflicts of interest to declare.