scholarly journals A Phase I Study of Allogeneic Natural Killer Cell Therapy Generated from Cord Blood Hematopoietic Stem and Progenitor Cells in Elderly Acute Myeloid Leukemia Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1357-1357 ◽  
Author(s):  
Harry Dolstra ◽  
Mieke W.H. Roeven ◽  
Jan Spanholtz ◽  
Basav Hangalapura ◽  
Marleen Tordoir ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Therapy ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Residual Disease ◽  
Standard Chemotherapy ◽  
Donor Chimerism ◽  
Elderly Aml

Abstract Introduction Elderly acute myeloid leukemia (AML) patients have a poor prognosis due to high relapse rates following standard therapy. Natural Killer (NK) cell alloreactivity has found to control relapse in AML in the HLA-mismatched haploidentical allogeneic stem cell transplantation (allo-SCT) setting. Moreover, allogeneic NK cell infusions can induce complete remission (CR) inpatients with advanced AML. As a consequence, adoptive NK cell transfer may be a promising treatment for elderly AML patients, who are not eligible for allo-SCT. Most clinical studies exploited NK cell products enriched from leukapheresis of haploidentical donors containing low numbers of T cells that could have contributed to the observed therapeutic effects and potentially induced graft-versus-host disease (GVHD). Therefore, we have developed a GMP-compliant culture system for the generation of large batches of NK cells from umbilical cord blood (UCB)-derived CD34+ progenitor cells, without T cell contamination. Here, we report results of a phase I dose escalation study (Dutch Trial Register nr. NTR2818) to evaluate the feasibility, safety and toxicity of allogeneic UCB-NK cell infusion following an immunosuppressive preparative regimen in elderly AML patients. Secondary endpoints were NK cell lifespan and the effects on minimal residual disease (MRD). Methods Elderly AML patients not eligible for allo-SCT, and in morphologic CR after standard therapy, were given preparative chemotherapy consisting of Cyclophosphamide (Cy;900 mg/m2/day) and Fludarabine (Flu;30 mg/m2/day) on days -6 to -2. At day 0, UCB-NK cells at a dose of 3, 10 or up to 30x106/kg body weight were infused without IL-2 treatment to study if in vivo expansion could be obtained without IL-2 support. Patients were assessed for toxicity and GVHD. Donor chimerism was measured by Q-PCR for discriminating DNA polymorphisms. NK cell expansion and phenotype were analyzed by flow cytometry. MRD was evaluated by flow cytometry and molecular techniques. Results Twelve AML patients (68-76 years) have been included, all in morphologic CR after 2 to 3 standard chemotherapy courses (n=6), or 1 standard chemotherapy course followed by subsequent treatment with hypomethylating agents (azacitidine or decitabine) (n=6). Patients were treated with Cy/Flu and an escalating dose of partially HLA-matched UCB-NK cells. Four patients had good/intermediate risk, 4 poor risk and 4 very poor risk AML. To date, 9 patients received NK cell products containing a median of 74% highly activated CD56+ NK cells, with <1x104/kg CD3+ T cells and <3x105/kg CD19+ B cells. Remaining non-NK cells were CD14+ and/or CD15+ monocytic and myelocytic cells. Follow up did not show GVHD or toxicity attributed to the NK cells. As expected, preparative Cy/Flu induced a neutropenic period of 20 ± 16 days, but no severe infections were seen. A temporary repopulation and persistence of UCB-NK cells could be detected in peripheral blood between days 1 and 8 post-infusion, which was associated with increased IL-15 plasma levels observed in most patients. Interestingly, donor chimerism increased with higher doses of infused UCB-NK cells, and donor chimerism up to 3.5% was found in bone marrow (BM) at day 7/8. Further UCB-NK cell maturation in vivo was observed by acquisition of CD16 and KIRs, while expression of activating receptors was sustained. Of the 9 treated patients so far, 5 (56%) are still in CR after 43, 35, 31, 5 and 4 months, whereas 4 patients relapsed after 5, 6 (2 pts) and 15 months. Despite morphologic CR during azacitidine treatment, residual disease of 6-7% with a leukemia-associated phenotype could be detected by flow cytometry before NK cell infusion in BM of two patients. In both patients MRD was reduced to less than 0.05% at 90 days after UCB-NK cell therapy following Flu/Cy conditioning. Conclusion These results show that GMP-compliant UCB-NK cell products containing up to 30x106 NK cells/kg body weight can be safely infused in non-transplant eligible AML patients following immunosuppressive chemotherapy. After infusion, UCB-NK cells repopulate, mature and migrate to BM without supporting IL-2 infusion. Since we observed reduction in MRD in patients on treatment with hypomethylating agents, this UCB-NK cell therapy may induce or sustain CR in elderly AML patients, and could serve as an alternative consolidation therapy for patients with refractory AML or provide bridge to allo-SCT. Disclosures Spanholtz: Glycostem Therapeutics: Employment. Tordoir:Glycostem Therapeutics: Employment. Bohme:Glycostem Therapeutics: Employment. Kok:Glycostem Therapeutics: Employment.

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 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

scholarly journals Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3607-3607
Author(s):  
Grace Lee ◽  
Sheela Karunanithi ◽  
Zachary Jackson ◽  
David Wald
Keyword(s):  
Cell Therapy ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Akt Activation ◽  
Nk Cell Cytotoxicity ◽  
Rock Inhibition ◽  
Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'&gt;0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii194-ii195
Author(s):  
Nazanin Majd ◽  
Maha Rizk ◽  
Solveig Ericson ◽  
Kris Grzegorzewski ◽  
Sharmila Koppisetti ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
In Vitro Cytotoxicity ◽  
Orthotopic Mouse Model ◽  
Hematopoietic Stem ◽  
Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

Transfer of Ex Vivo Expanded NK and γδT Cells from Untouched Posttransplant PBMCs to Clear Minimal Residual Disease in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5294-5294
Author(s):  
Patrick Schlegel ◽  
Chihab Klose ◽  
Christina Kyzirakos ◽  
Ursula J.E. Seidel ◽  
Kai Witte ◽  
...  
Keyword(s):  
Cell Culture ◽  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Cell Expansion ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Residual Disease ◽  
Cell Transfer ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract GMP-grade NK cell expansion for clinical purpose has been demonstrated feasible and safe. Here we share our pilot data on posttransplant immunotherapy with ex vivo expanded NK cells to treat minimal residual disease in a pediatric patient with posttransplant relapsed myeloid leukemia. Our patient, a 13 year old boy who underwent 2nd allogeneic stem cell transplantation (haploidentical stem cell transplantation from his mother) due to posttransplant relapsed acute myeloid leukemia. After the 2nd haploidentical stem cell transplantation (SCT) minimal residual disease (MRD) was detected by multiparameter flow cytometry and by two molecular markers CALM-AF10 fusion transcript and a NRAS-mutation. For posttransplant compassionate use immunotherapy by NK cell transfer, NK cells were expanded from untouched isolated PBMCs of the patient post 2nd haploidentical SCT. GMP-grade expansion of the NK cells was done under static conditions in our GMP-facility. Isolated PBMCs were pooled with 100Gy irradiated K562mb15 4-1BBL feeder cells (kindly provided by Dario Campana) in a proportion of 1:20 (NK to K562mb15 4-1BBL). PBMCs and K562mb15 4-1BBL were seeded in conventional cell culture flasks (175cm2) at a density of 1.1E6 cells/ml. Cell culture media contained RPMI1640 supplemented with 10% AB-human serum, 1% L-glutamine and 100IU Proleukine® IL2/ml. Cell culture was monitored daily for cell number, white blood cell differentiation, pH of the cell culture, glucose metabolism, lactate production and microbial sterility testing at the beginning and the end of the expansion period. The cell product was harvested on day 15-17. Fresh isolated PBMCs and the expanded NK cell product were characterized by flow cytometry. NK cells were expanded &gt;1000 fold (3.1 and 3.4 log-fold) in 14-17 days. The product contained a total number of 9.8E9 and 19.9E9 cells, which was 328 and 665E6/kgBW. The expansion protocol supports NK and γδ T cell expansion whereas the number of αβ T cells stays stable. Cytotoxicity assay against various targets revealed excellent cellular cytotoxicity and antibody dependent cellular cytotoxicity. To prevent relapse in our patient with posttransplant MRD positivity, NK cells from the patient post 2nd haploidentical SCT were expanded for cellular immunotherapy. 2 weeks post 1st NK cell transfer (day +170) the patient achieved complete MRD response in the bone marrow. Unfortunately the patient showed detectable MRD one month later. Therefore another NK cell expansion and transfer was done. 2 weeks post 2nd NK cell transfer (day +232) the patient again achieved complete MRD response in the bone marrow and is in complete molecular remission ever since (day +340). The NK cell products were tolerated well. Transient coughing and temporary increase of temperature were registered. Both, in vitro and in vivo effect of the NK cell product were documented. Clinical use of expanded and activated NK cells and γδ T cells can induce molecular remission in posttransplant MRD positive acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients

Blood ◽  
2011 ◽  
Vol 118 (12) ◽  
pp. 3273-3279 ◽  
Author(s):  
Antonio Curti ◽  
Loredana Ruggeri ◽  
Alessandra D'Addio ◽  
Andrea Bontadini ◽  
Elisa Dan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Nk Cells ◽  
Natural Killer ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Median Number ◽  
Active Disease ◽  
Acute Myeloid

Abstract Thirteen patients with acute myeloid leukemia, 5 with active disease, 2 in molecular relapse, and 6 in morphologic complete remission (CR; median age, 62 years; range, 53-73 years) received highly purified CD56+CD3− natural killer (NK) cells from haploidentical killer immunoglobulin-like receptor–ligand mismatched donors after fludarabine/cyclophosphamide immunosuppressive chemotherapy, followed by IL-2. The median number of infused NK cells was 2.74 × 106/Kg. T cells were < 105/Kg. No NK cell–related toxicity, including GVHD, was observed. One of the 5 patients with active disease achieved transient CR, whereas 4 of 5 patients had no clinical benefit. Both patients in molecular relapse achieved CR that lasted for 9 and 4 months, respectively. Three of 6 patients in CR are disease free after 34, 32, and 18 months. After infusion, donor NK cells were found in the peripheral blood of all evaluable patients (peak value on day 10). They were also detected in BM in some cases. Donor-versus-recipient alloreactive NK cells were shown in vivo by the detection of donor-derived NK clones that killed recipient's targets. Adoptively transferred NK cells were alloreactive against recipient's cells, including leukemia. In conclusion, infusion of purified NK cells is feasible in elderly patients with high-risk acute myeloid leukemia. This trial was registered at www.clinicaltrial.gov as NCT00799799.

scholarly journals Human Cytokine-Induced Memory-like NK Cells Exhibit in Vivo Anti-Leukemia Activity in Xenografted NSG Mice and in Patients with Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 101-101
Author(s):  
Rizwan Romee ◽  
Rosario Maximillian ◽  
Melissa M Berrien-Elliott ◽  
Julia A Wagner ◽  
Brea A Jewell ◽  
...  
Keyword(s):  
Nk Cells ◽  
Myeloid Leukemia ◽  
Low Dose ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Cell Frequency ◽  
Nsg Mice ◽  
Post Infusion

Abstract Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia. Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P<0.001), following 7 days of rest in low dose IL-15 vitro. In addition, memory-like NK cells had increased granzyme B expression (P<0.01), and enhanced killing of K562 leukemia targets in vitro (P<0.05). Utilizing an in vivo xenograft model of human NK cells in NSG mice (Leong J et. al., BBMT, 2014), IL-12/15/18-induced memory-like NK cells that differentiated in NSG mice for 7 days exhibited increased IFN-g responses after ex vivo re-stimulation with K562 leukemia, confirming their memory-like functionality (P<0.05). To test in vivo responses to human leukemia in this model, luciferase-expressing K562 cells were engrafted into NSG mice (1x106/mouse, IV), and on day 3, groups of mice were injected with IL-12/15/18-pre-activated or control NK cells from the same donor (4x106/mouse). Mice treated with a single dose of memory-like NK cells exhibited significantly improved in vivo leukemia control measured by whole mouse bioluminescent imaging (P=0.03), as well as overall survival (P<0.05), compared to mice treated with control or no NK cells. Based on these pre-clinical findings, we initiated a first-in-human clinical trial of HLA-haploidentical IL-12/15/18-induced memory-like NK cells in patients with AML (NCT01898793). Relapsed/refractory (rel/ref) AML patients receive lymphodepleting non-myeloablative flu/cy conditioning, infusion of a single dose of CD56+CD3- memory-like donor NK cells, followed by two weeks of low dose rhIL-2. Three patients were treated at dose level 1 (0.5x106 cells/kg) and two patients treated at dose level 2 (1.0x106/kg) with no DLTs observed, and accrual continues. Correlative analyses utilizing donor-specific HLA mAbs allow tracking of donor memory-like NK cell frequency and function following adoptive transfer. Donor memory-like NK cells were detectable in the PB and BM of all tested patients with informative HLA (4/5), peak in frequency at 7-8 days post-infusion, and contract after 14-21 days as expected following recipient T cell recovery (Figure). Memory-like NK cells exhibit significantly increased Ki67%+ as a marker of proliferation at day 7 [97.8+1.0% (donor) vs. 21.6+5.5% (recipient), mean+SEM, P<0.001]. Moreover, functional analyses of NK cells at days 7-8 post-infusion reveal increased numbers of donor IFN-g+ NK cells following restimulation with K562 leukemia cells in the same blood [1009+590 (donor) vs. 8+3 (recipient) IFN-g+ NK cells] or BM [686+423 (donor) vs. 4+2 (recipient) IFN-g+ NK cells] samples. Two of four evaluable patients treated with memory-like NK cells had leukemia free BM and PB at days 14 post-therapy, which correlated with BM NK cell frequency and IFN-g production (Figure). CIML007 had rel/ref AML with 48% BM blasts pre-therapy, and had no evidence of leukemia on day 14, 28, and 100 BM biopsies, and has an ongoing complete remission more than 100 days after this therapy. CIML009 had 80% BM blasts pre-therapy, and had no evidence of leukemia on day 14 BM biopsy post-infusion. Thus, human IL-12/15/18-induced memory-like NK cells expand and have enhanced anti-AML function following adoptive transfer in patients, thereby constituting a promising translational innovation for immunotherapy of AML. Figure 1. Figure 1. Disclosures Fehniger: Celgene: Research Funding.

siRNA Inactivation of the Inhibitory Receptor NKG2A Augments the Anti-Tumor Effects of Adoptively Transferred NK Cells In Tumor-Bearing Hosts

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1015-1015 ◽  
Author(s):  
Elissa Furutani ◽  
Su Su ◽  
Aleah Smith ◽  
Maria Berg ◽  
Richard Childs
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Nk Cell ◽  
Tumor Burden ◽  
Cell Cytotoxicity ◽  
Inhibitory Receptor ◽  
Inhibitory Receptors ◽  
Tumor Bearing ◽  
Nk Cell Cytotoxicity

Abstract Abstract 1015 Natural killer (NK) cells are a component of the innate immune system that target both tumors and virally infected cells. NK cell killing of tumors is regulated by a delicate balance of activating and inhibitory receptors. These inhibitory receptors bind HLA ligands which prevent NK cell targeting of normal “self” cells. Up regulation of surface expression of HLA molecules has been utilized by tumors as a method to evade NK cell cytotoxicity. Disrupting the function or expression of inhibitory receptors on NK cells could potentially be used as a method to overcome this effect. While most inhibitory receptors are present in only a subset of NK cells, NK cells universally express the HLA-E binding inhibitory receptor NKG2A. We hypothesized that siRNA inactivation of NK cell NKG2A would could be used as a therapeutic approach to enhance NK cell tumor cytotoxicity in vivo. The human natural killer cell line NKL was transduced with lentiviral vectors encoding shRNA targeting various regions of the NKG2A transcript. Following lentiviral transduction, knockdown of receptor expression was confirmed by flow cytometry and RT-qPCR. Compared to wild type (WT) and GFP-transduced NKL controls, NKG2A silenced NKL cells had increased secretion of IFN-gamma and Fas-L by ELISA and increased granzymes A and B and Nkp30 expression by flow cytometry. In contrast, expression of NKG2D, Nkp44, Nkp46, LFA-1, DNAM, and TRAIL was not altered by NKG2A silencing. Chromium-based cytotoxicity assays showed shRNA knockdown of NKG2A significantly enhanced NK cell cytotoxicity of tumor cells: at a 20:1 effector to target ratio, NKG2A knockdown NKLs, WT NKLs and GFP-transduced NKLs induced 68.9%, 8.2% and 8.3% lysis respectively of 721.221 EBV-LCL tumor targets (p=0.001). Remarkably, NKG2A silencing enhanced NKL killing of both HLA-E positive (721.221 EBV-LCL and 526 melanoma cells) and HLA-E negative (K562) tumor cell lines, suggesting NKG2A inactivation increased NK cell cytotoxicity through both HLA-E dependent and independent mechanisms. Using a xenogeneic model, we next explored the in vivo effects of transferring NKG2A silenced NK cells in tumor bearing mice. Immunodeficient NSG mice were injected with 1 million human luciferase transduced 721.221 HLA-E expressing EBV-LCL tumor cells. Twenty-four hours later, tumor-bearing mice were injected with 2–5 million WT NKL cells, GFP-control-transduced NKL, or NKG2A silenced NKL cells, then received IL-2 sq for 10 days to induce in vivo NK cell proliferation. NKL numbers in blood were subsequently analyzed by flow cytometry and tumor burden was assessed by luciferase-based bioluminescence imaging (BLI). At 16 and 21 days following adoptive NK cell transfer, BLI showed that recipients of NKG2A silenced NKL cells had slower tumor growth and significantly smaller tumor burden compared to NKL wt and NKL-GFP transduced controls (figure). Importantly, no toxicity related to infusing NKG2A inactivated NK cells was observed. These in vitro and in vivo data suggest shRNA knockdown of the NKG2A inhibitory receptor could be used as a method to augment NK cell tumor cytotoxicity in patients with hematological malignancies. Figure: Tumor burden in mice Luciferase-tagged 721.221 HLA-E EBV LCLs were injected into mice and imaged using a bioluminescence imager at days 10, 16, and 22 following NKL injection. 5 mice were followed in each group. Figure:. Tumor burden in mice . / Luciferase-tagged 721.221 HLA-E EBV LCLs were injected into mice and imaged using a bioluminescence imager at days 10, 16, and 22 following NKL injection. 5 mice were followed in each group. Disclosures: No relevant conflicts of interest to declare.

Induction of NK Cell Reactivity Against Myeloid Leukemia By a Novel Fc-Optimized CD133 Antibody

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3793-3793 ◽  
Author(s):  
Kathrin Rothfelder ◽  
Samuel Koerner ◽  
Maya Andre ◽  
Julia Leibold ◽  
Philaretos Kousis ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

Abstract NK cells largely contribute to the success of monoclonal antibody (mAb) application in cancer due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), a feature considered critical for therapeutic success. Up to now, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Recently, we reported on the development of mAb targeting CD133, which is expressed on a wide variety of tumor cells (Koerner et al., Blood 2014 124:2309). Here we extend our analyses and provide further data on the preclinical characterization of an Fc-engineered CD133 mAb for the treatment of myeloid leukemia. Compared to two other anti-human CD133 mAb (clones AC133 and W6B3), which both bound to primary AML and CML cells in 15/25 and 7/10 cases, respectively, clone 293C3 recognized the leukemic cells in 22/25 AML cases and 7/10 CML cases. Based on these results, clone 293C3 was chosen to generate chimeric mAb with either a wildtype Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance affinity to the activating Fc receptor CD16 on NK cells (293C3-SDIE). Treatment with 293C3-SDIE resulted in significantly enhanced activation, degranulation and lysis of primary CD133-positive AML cells by NK cells in allogeneic and autologous experimental ex vivo settings as compared to its wildtype counterpart. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow (BM) cells. In line with the observed lower expression levels of CD133 on healthy compared to malignant hematopoietic cells no relevant toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells was observed. Moreover, 293C3-SDIE did not induce lysis of of healthy BM cells by allogeneic or autologous NK cells. In a NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) xenotransplantation model, induction of ADCC by treatment with 293C3-SDIE resulted in the elimination of patient AML cells by NK cells from a matched human donor. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound, in particular for the elimination of minimal residual disease in CD133 bearing leukemia in the context of allogenic SCT. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document