In Vitro Expanded NK Cells Have Increased Natural Cytotoxity Receptors, TRAIL and NKG2D Expression, and Superior Tumor Cytotoxicity Compared to Short-Term IL-2 –Activated NK Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 463-463 ◽  
Author(s):  
Maria Berg ◽  
Andreas Lundqvist ◽  
Dawn Betters ◽  
Richard W. Childs
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Natural Cytotoxicity ◽  
Short Term ◽  
Tumor Cytotoxicity ◽  
Natural Cytotoxicity Receptors

Abstract Abstract 463 IL-2 activates NK cell enhancing their capacity to lyse tumor cells. To date, most clinical studies of adoptive NK cell transfer have utilized short-term (12-16 hours) IL-2-activated NK cells. Because IL-2 alone is ineffective in expanding NK cells in vitro, the relatively low numbers of NK cells obtained for infusion following short term IL-2 activation may limit the full therapeutic impact of this approach. To obtain larger numbers of NK cells, novel ex vivo expansion protocols that utilize irradiated EBV-LCL or K562 feeder cells have recently been developed. However, concerns exist that extensive ex vivo expansion might significantly reduce the in vivo proliferative potential and long-term viability of adoptively transferred NK cells. Here we investigated for differences in phenotype, tumor cytotoxicity and in vivo persistence between short-term IL-2 activated and long-term expanded NK cells. CD56+/CD3- NK cells were isolated from normal donors by immuno-magnetic bead selection and were either activated with IL-2 (500U/ml) for 12-16 hours or were expanded in vitro over 14 days using irradiated EBV-LCL feeder cells in IL-2 containing media (500U/ml). Short-term IL-2 activated NK cells did not expand in number in contrast to EBV-LCL stimulated NK cells which expanded 400-1000 fold by culture day 14. Flow cytometry analysis revealed no differences in expression of DNAM-1, 2B4, LFA-1 or granzyme B between short-term activated and expanded NK cells. However, expanded NK cells had significantly higher expression of TRAIL, NKG2D, and the natural cytotoxicity receptors NKp30, NKp44 and NKp46 and a slight increase in KIR3DL1 and KIR2DL2/3. A 4-hour 51Cr-release assay showed expanded NK cells were significantly more cytotoxic against K562 cell compared to short-term IL-2 activated NK cells; at a 1:1 effector to target ratio, 67±6%, 26±1%, and 9±1% of K562 cells were killed by expanded, short term IL-2 activated and fresh NK cells respectively (p<0.05). Increased TRAIL expression on expanded NK cells was also associated with increased lysis of TRAIL-sensitive tumor cells (RCC tumors treated with bortezomib); at a 1:1 E:T ratio, 55±3% and 5±2% of bortezomib-treated RCC tumors were killed by expanded and short-term IL-2 activated NK cells respectively (p<0.05). We next assessed for differences in the in vivo longevity of these NK cell populations when transferred into immuno-deficient mice. Two million NK cells were labeled with a near infrared-dye (DiR; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) and injected intra-peritoneal (i.p.) into CB.17 SCID-beige mice. Mice were administered IL-2 (100,000U/ml bid i.p.) for five days then underwent bioluminescent imaging using the IVIS100 system. Although FACS analysis of NK cells performed immediately prior to injection showed increased DiR fluorescent intensity in short-term IL-2 activated vs. expanded NK cells, fluorescence signal in vivo was slightly higher in the first 24-96 hours in mice that received expanded NK cells; fluorescence intensity was 5-41% (p=0.003) stronger in recipients of expanded NK cells compared to mice receiving short-term IL-2 activated NK cells. We next evaluated the in vivo anti-tumor effects of activated vs. expanded NK cells. CB.17 SCID-beige mice were injected i.p. with luciferase transduced 526 human melanoma cells three days prior to receiving an i.p. injection of short term IL-2 activated vs. expanded NK cells (+ bid i.p. IL-2). Bioluminescent imaging measuring tumor flux to calculate tumor burden and tumor doubling time showed no difference in tumor progression between both NK cell cohorts. In conclusion, these results demonstrate that ex vivo expanded NK cells are phenotypically and functionally different than short-term IL-2 activated NK cells. Expanded NK cells have increased expression of natural cytotoxicity receptors, NKG2D and TRAIL and have greater TRAIL-mediated tumor cytotoxicity compared to IL-2 activated NK cells. Importantly, despite extensive ex vivo proliferation, expanded NK cells appear maintain similar longevity in vivo as non-expanded short term IL-2 activated NK cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Paul D. Bates ◽  
Alexander L. Rakhmilevich ◽  
Monica M. Cho ◽  
Myriam N. Bouchlaka ◽  
Seema L. Rao ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

scholarly journals 852 Trifunctional NKp46/CD16a-NK cell engager targeting CD123 overcomes acute myeloid leukemia resistance to ADCC

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A893-A893
Author(s):  
Laurent Gauthier ◽  
Angela Virone-Oddos ◽  
Angela Virone-Oddos ◽  
Jochen Beninga ◽  
Benjamin Rossi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Antitumor Activity ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Activating Receptors ◽  
Acute Myeloid

BackgroundThere is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.1 However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.2 T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.3 Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.4MethodsWe report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp464 and CD123.5 We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies.ResultsThe expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release.ConclusionsThe data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development.ReferencesEhninger A, Kramer M, Rollig C, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J 2014;4:e218.Montesinos P, Gail J Roboz GJ, et al. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. Leukemia 2021;35(1):62–74.Uy GL, Aldoss I, Foster MC, et al. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. Blood 2021;137(6):751–762.Gauthier L, Morel A, Anceriz N, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13.Jin L, Lee EM, Ramshaw HS, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3928-3928
Author(s):  
Michele Levin ◽  
Janet Ayello ◽  
Frances Zhao ◽  
Andrew Stier ◽  
Lauren Tiffen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Scid Mice ◽  
Activation Marker

Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 580-580
Author(s):  
Mark Wunderlich ◽  
Mahesh Shrestha ◽  
Lin Kang ◽  
Eric Law ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Cell Product ◽  
Equity Ownership ◽  
Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

scholarly journals Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 341-341
Author(s):  
Lucila Kerbauy ◽  
Mecit Kaplan ◽  
Pinaki P Banerjee ◽  
Francesca Lorraine Wei Inng Lim ◽  
Ana Karen Nunes Cortes ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

Treatment of Ex Vivo Expanded NK Cells with Daratumumab F(ab')2 Fragments Protects Adoptively Transferred NK Cells from Daratumumab-Mediated Killing and Augments Daratumumab-Induced Antibody Dependent Cellular Toxicity (ADCC) of Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4244-4244 ◽  
Author(s):  
Elena Cherkasova ◽  
Luis Espinoza ◽  
Ritesh Kotecha ◽  
Robert N. Reger ◽  
Maria Berg ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Killing ◽  
Cellular Toxicity ◽  
Myeloma Cells ◽  
Nsg Mice

Abstract Daratumumab is a fully humanized monoclonal antibody (IgG1) that targets CD38 expressed on myeloma cells. Daratumumab kills myeloma cells through antibody dependent cellular toxicity (ADCC), compliment dependent cytotoxicity (CDC), and antibody dependent phagocytosis (ADCP). In early clinical trials, daratumumab has showed significant anti-myeloma activity in patients with treatment refractory disease. In vivo, daratumumab has been found to induce NK cell lymphopenia of unclear etiology. We found that NK cells isolated from the peripheral blood of healthy and cancer patients expressed variable surface levels of CD38 (Fig. 1A). Further, surface expression of CD38 increased substantially when NK cells underwent ex vivo cytokine activation by culturing cells overnight in IL-2 containing media or ex vivo expansion using irradiated EBV-LCL feeder cells (Fig. 1B). Remarkably, daratumumab induced apoptosis of expanded NK cells in a dose dependent manner, with substantial NK cell apoptosis occurring within 2 hours following in vitro exposure to daratumumab at a concentration of 1 and 10 ug/ml (Fig. 1C). Further, adoptive transfer of ex vivo expanded human NK cells into NSG mice that had been pre-treated with daratumumab showed daratumumab induced NK cell killing in vivo: the numbers of NK cells isolated from the lungs, blood, spleen and bone marrow of NSG mice 24 hours after infusion of expanded human NK cells was reduced by 90% in mice that were pretreated with 1 mg/kg of daratumumab i.p. compared to controls that had not received the antibody (Fig. 1D). In vitro experiments showed NK cell killing by daratumumab occurred as a consequence of ADCC and was dependent on NK cell CD16 expression; when CD56+ NK cells were sorted by FACS into CD16 positive and negative populations, only NK cells expressing CD16 were killed by daratumumab, with no effect on NK cell viability occurring in the CD16- NK cell. Further, we observed that NK cells obtained from donors who have high affinity FCgR3 as a consequence of a single nucleotide polymorphism in the FCGR3A gene resulting in an amino acid substitution at position 158 (F158V) in CD16 were more sensitive to daratumumab killing compared to NK cells isolated from donors carrying the low affinity CD16 polymorphism. Although NK cell counts and NK reduction in peripheral blood and bone marrow were not associated with daratumumab clinical response in myeloma studies, NK cells play an important role in mediating antitumor responses through ADCC following mAb therapy. In this regard, combining mAb therapy with adoptive transfer of ex vivo expanded NK cells could be utilized as a strategy to potentiate the antitumor effects of mAbs. To overcome daratumumab-mediated killing of adoptively transferred NK cells in daratumumab-treated patients, we blocked CD38 on the surface of NK cells by pretreating them with daratumumab F(ab')2 fragments. The F(ab')2 fragments that were generated using pepsin cleavage of daratumumab were confirmed to bind and block the CD38 epitope expressed on NK cells. Importantly, these F(ab')2 fragments remained bound to the surface of NK cells for at least 96 hours, did not induce NK cell apoptosis, protected NK cells from daratumumab-mediated NK cell killing, and bolstered their tumor cytotoxicity against daratumumab-treated myeloma targets. In vitro experiments showed NK cell tumor cytotoxicity vs myeloma cells in daratumumab-containing media was significantly higher by NK cells that had CD38 blocked with F(ab')2 fragments compared to unblocked controls (Fig. 1E). Importantly, pretreatment with daratumumab F(ab')2 fragments also protected human NK cells from daratumumab-mediated killing in vivo; expanded NK cells pretreated with F(ab')2 fragments prior to adoptive transfer into NSG mice that had been treated with daratumumab were detectable at significantly higher numbers in the blood compared to untreated NK cell controls (Fig. 1F). Conclusion: Expression of CD38 on activated NK cells makes them susceptible to killing by daratumumab, which could compromise the ability of adoptively transferred NK cells to bolster ADCC following treatment with this mAb. Pretreatment of ex vivo expanded NK cells with daratumumab F(ab')2 fragments protects cells from daratumumab-mediated killing, potentially offering a strategy to augment the anti-tumor effects of adoptively transferred NK cells in myeloma patients that have received daratumumab treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 194-194
Author(s):  
Pamela Wong ◽  
Carly C. Neal ◽  
Lily Chang ◽  
Julia A Wagner ◽  
Melissa M. Berrien-Elliott ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
T Box ◽  
Nsg Mice ◽  
Healthy Donors ◽  
And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p &lt; 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (&lt;30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p&lt;0.01, Figure C), blood (6-fold decrease, p&lt;0.01), and liver (4-fold decrease, p&lt;0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p&lt;0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p&lt;0.05), and IFN-γ production was reduced (p&lt;0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

138 In vivo localization of genetically engineered natural killer cells against glioblastoma using PET imaging

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A151-A151
Author(s):  
Yeonhee Yun ◽  
Jiao Wang ◽  
Karen Pollok ◽  
Tony Sinn ◽  
Randy Brutkiewicz ◽  
...  
Keyword(s):  
Mr Imaging ◽  
Nk Cells ◽  
Natural Killer ◽  
Pet Imaging ◽  
Nk Cell ◽  
Ex Vivo ◽  
Killing Activity ◽  
Gamma Counting

BackgroundGlioblastoma (GBM) is a deadly brain malignancy with a dismal prognosis. While immunotherapy holds great promise for GBM treatment, most have failed due to a suppressive tumor microenvironment (TME). Antigen heterogeneity and adenosine signaling are two immunosuppressive mechanisms in GBM. The CD73-adenosine axis plays a multifaceted role in GBM pathogenesis and drives the dysfunction of NK cells in GBM TME.1,3 Our NKG2D-chimeric antigen receptor (CAR)-natural killer (NK) cells have shown anti-tumor activity when combined with CD73 blockade in vivo.2 To further extend the potency of these cells against GBM and address antigen heterogeneity in GBM, we combined the local blockade of CD73 with multi-antigen-targeting engineered NK cells. In order to improve treatment assessment, PET/MR imaging was employed to enable detailed, non-invasive assessment of tumor progression. Imaging assessment of adoptively-transferred CAR- NK cells was also developed to determine the fate of NK cell delivery to the tumor site over time.MethodsWe generated multifunctional engineered NK (E-NK) cells that express an anti-CD73 scFv, which is cleavable by GBM-associated proteases, an NKG2D-CAR, as well as a GD2 CAR, which can actively target the GD2 antigen overexpressed on GBM (Figure 1A). For E-NK cell radiolabeling, zirconium-89 (89Zr, ½ life = 78 Hr) radiotracer was attached covalently to the E-NK cell surface via conjugation with DFO-Bz-NCS in a range of doses from 50–600 µCi.ResultsAn optimal balance between labeling efficiency and cell viability was attained at 120 µCi 89Zr resulting in 39% labeling efficiency and 46% cell viability over for 48 hours. After labeling, the NK cells maintained their in vitro killing activity against GBM cells (figure 1B). The 89Zr labeled E-NK cells were administered intravenously in mice containing intracranial GBM10 tumors at week 5 post-implant. PET imaging was performed at 1 and 2 days later and gamma imaging ex vivo at 4 days. Free 89Zr was visible diffusely throughout the body with low levels in the brain. The majority of 89Zr labeled E-NK cell groups localized to the lungs with detectable activity elsewhere in various organs (figure 1C and 1D).Abstract 138 Figure 1PET imaging and gamma counting of the engineered NK cellsFigure 1 (A) Multifunctional, responsive CAR constructs; (B) In vitro killing activity against GBM43 cells after co-incubation with 89Zr labeled NK cells at an E:T ratio of 10 for 4 h with LDH assay (N=3); (C) & (D) In vivo PET imaging and ex vivo gamma counting with 89Zr at week 5 in 10 mice during 4 days, GBM intracranial implantation to NSG male mouse, 89Zr, 89Zr + NK cell, or 89Zr + E NK cell (7 × 106 cells with 500 µCi) was administered through intravenous injection, Qimage was used for the PET/MRI co-registration and analysisConclusionsWe generated multifunctional E-NK cells which showed the improved killing of GBM cells using novel targeting approaches, including the blockade of CD73-mediated adenosinergic signaling. We also optimized E-NK cell radiolabeling with 89Zr for GB10 therapy in vitro and in vivo fate mapping against a xenograft of patient-derived GBM.AcknowledgementsWe gratefully acknowledge the Walther Oncology Embedding Program, Indiana University Simon Cancer Center, and In Vivo Therapeutics Core.ReferencesWang J, Matosevic S. NT5E/CD73 as correlative factor of patient survival and natural killer cell infiltration in glioblastoma. J Clin Med 2019;8(10):1526.Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunother Cancer 2018;6(1):136.Yan A, Joachims ML, Thompson LF, Miller AD, Canoll PD, Bynoe MS. CD73 promotes glioblastoma pathogenesis and enhances its chemoresistance via A2B adenosine receptor signaling. J Neurosci 2019;39(22):4387.Flink J, Muzi M, Peck M, Krohn K. Multimodality brain tumor imaging: mr imaging, PET, and PET/MR imaging. J Nucl 2015;5(10):1554–1561.

Anti-KIR (1-7F9): A Fully Human Monoclonal Antibody (mAb) That Blocks KIR2DL1, −2 and −3, Promoting Natural Killer (NK) Cell-Mediated Lysis of Tumor Cells In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 582-582 ◽  
Author(s):  
Nicolai Wagtmann ◽  
Pascale Andre ◽  
Stefan Zahn ◽  
Pieter Spee ◽  
Nicolas Anfossi ◽  
...  
Keyword(s):  
Nk Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Phase 1 ◽  
Human Monoclonal Antibody ◽  
Hla Class I ◽  
Term Survival

Abstract In patients with acute myeloid leukemia (AML), haplo-identical stem cell transplantation (SCT) can lead to expansion and activation of Killer Immunoglobulin-like Receptor (KIR)-HLA class I mismatched NK cells, resulting in reduced rates of leukemia relapse and no graft-versus-host disease (Ruggeri et al. Science 2002). However, this SCT is not available to the majority of AML patients who are elderly. To explore the feasibility of achieving similar NK-mediated anti-leukemia activity by a pharmacological approach, we generated fully human anti-KIR mAbs that block the interactions of inhibitory KIR2DL receptors with their HLA-C ligands, thereby enhancing NK activity. Here we describe one such therapeutic candidate anti-KIR mAb, designated 1-7F9. As distinct HLA-C allotypes are recognized by KIR2DL1 or −2/3, only mAbs that cross-react with these KIRs would be expected to work in the entire population. Hence, 1-7F9 was initially selected based on its ability to bind soluble, recombinant KIR2L1, −2 and −3. By Biacore analysis, the bivalent affinities for KIR2DL1 and −3 were 0.43 × 10−9 M and 0.025 × 10−9 M, respectively. In experimental systems and in normal human blood, 1-7F9 bound KIR2DL1, −2 and −3, and −2DS1 and −2, but not to KIR2DS3 or −4. 1-7F9 dose-dependently inhibited the binding of soluble KIR2DL1-Fc to cell surface HLA-Cw4. 1-7F9 augmented the lysis of 721.221-Cw4 B-EBV cells by an NK cell line transfected with KIR2DL1 (YTS-2DL1) from 5% lysis in absence of mAb to a maximal 55% lysis at 5 ug/ml of mAb, but did not affect lysis by KIR-negative NK cells. Lysis of PHA-stimulated blasts and primary AML blasts by autologous IL-2 activated NK cells (E:T=6:1) was 10 and 15%, respectively, in absence of mAb vs 80% and 55% in presence of 1-7F9. Incubation of IL-2 activated blood mononuclear cells with 1-7F9 resulted in expression of the activation marker CD107 on about 10% of KIR2D-positive NK cells, which increased to 20% upon addition of HLA-C-positive B-EBV targets, suggesting that 1-7F9 preferentially induces activation of NK cells in presence of transformed cells. The isotype of 1-7F9 is IgG4; accordingly, it did not cause depletion of KIR positive cells in vitro or in vivo in KIR-transgenic mice despite long-lived KIR-occupancy. As KIR are not found in mice, in vivo activity was tested in a NOD-SCID mouse model where inoculation of in vitro-expanded NK cells (80% of NK cells KIR2D-positive) and autologous human B-EBV cells (E:T=1:3) resulted in death of all mice by day 26. A single injection of 1-7F9 (125 ug/mouse) resulted in long-term survival, with 100% of treated mice alive beyond day 60; in contrast, 60 ug/mouse of the mAb was ineffective. Similarly, ex vivo pre-incubation of NK cells with 1-7F9 (37,3 ug/106 NK cells) prior to inoculation in mice resulted in elimination of the autologous transformed B cells in vivo and survival of 100% of the treated animals. These data show that 1-7F9 augments NK-mediated tumor killing in vitro and in vivo, and that it exhibits long-lived KIR binding in vivo, providing a preclinical basis for initiating phase 1 clinical trials with the mAb.

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