Expanded Natural Killer Cells For The Control Of Minimal Residual Disease In Ph+ Acute Lymphoblastic Leukemia Patients

Abstract The management of Ph+ acute lymphoblastic leukemia (ALL) patients has profoundly changed during the last decade. Virtually all adult Ph+ ALL patients, including the elderly where the abnormality accounts for over 50% of cases, can obtain a complete remission (CR) with the use of TK inhibitors (and steroids) without systemic chemotherapy (Vignetti M et al. Blood 2007;109:3676; Foà R et al. Blood 2011;118:6521). Most patients remain, however, minimal residual disease (MRD)-positive. The possibility of targeting MRD via an immune-mediated control is particularly appealing in these patients. Previous studies have shown that natural killer (NK) cells with killing activity against autologous blasts may be expanded from ALL patients in CR (Torelli GF et al. Haematologica 2005;90:785). NK cell recognition of malignant targets is regulated by activating and inhibitory receptors. The major receptors with activating functions are NKG2D, DNAM-1 and the natural cytotoxicity receptors (NCRs) (NKp30, NKp44 and NKp46). MIC-A/B and ULBPs are ligands for NKG2D, PVR and Nec-2 for DNAM-1, while NCRs are orphan receptors. The pathways of NK-ALL recognition are unclear. Possible differences in NK cell killing susceptibility and in the expression of NK cell activating ligands among subgroups of ALL patients have been suggested. The aims of this study were: 1) to analyze the pathways of NK-ALL recognition, with particular attention to Ph+ samples; and 2) to verify whether differences in NK cell activating receptor ligand expression among molecularly-defined subgroups of patients correlate with the susceptibility to recognition and killing by NK cells activated and expanded under GMP conditions. PBMCs were collected from 23 healthy donors and 3 adult Ph+ ALL patients in CR. NK cells were enriched and cultured for 14 days in the presence of irradiated autologous feeder cells, autologous plasma, IL-2 and IL-15. The expression of the activating receptors NKG2D, DNAM-1 and NCRs was then analyzed. Samples from 46 newly diagnosed adult ALLs, median age 34 years (18-74), were also investigated: 39 patients had B-ALL - 15 BCR-ABL+, 7 MLL-AF4+, 2 E2A-PBX1+, 15 negative - and 7 had T-ALL. The expression of the NKG2D and DNAM-1 ligands on ALL blasts was analyzed. The cytotoxic activity of ex vivo expanded NK cells against primary ALL blasts was determined in a 51Cr release assay. For blocking experiments, NK cells were pre-treated with the anti-NKG2D or anti-DNAM-1 neutralizing mAbs. NK cells from healthy donors and from Ph+ ALL patients could be expanded respectively 33.2±15.2 and 39.1±19.3 fold. Expanded NK cells were represented by a homogenous population displaying a high expression of CD56 and CD16, in the absence of CD3. DNAM-1, NKG2D, NKp30 and NKp44 activating receptors presented a significantly increased expression after expansion from healthy donors (DNAM-1 p=.0007; NKG2D p=.0004; NKp30 p=.05; NKp44 p=.001). DNAM-1 and NKG2D showed a significantly increased expression after expansion also from Ph+ ALL patients (DNAM-1 p=.0012; NKG2D p=.045), while the expression of NCRs was not tested on these samples. The phenotypic analysis performed within molecularly-defined subgroups of ALL revealed that Ph+ cases presented an overall high surface expression of NKG2D and DNAM1 ligands. In particular, when compared to ALLs carrying no known molecular markers, Ph+ samples showed significantly higher levels of ULBP-1, ULBP-3 and MIC-B (p=.008, p=.026 and p=.033, respectively). In line with the phenotypic results, primary blasts from Ph+ ALL (n=5) appeared significantly more susceptible to NK-dependent lysis than B-ALL without molecular aberrations (n=6) (p=.007). When cytotoxic assays were performed in the presence of neutralizing mAbs, the NK cell killing potential was significantly inhibited by anti-DNAM-1 (p=.006), suggesting a pathway of recognition of ALL blast cells in the setting of the Nec-2/DNAM-1 interaction. The high expression of ligands for activating receptors in Ph+ ALL cases, together with the highest levels of susceptibility to NK cell-mediated lysis by this subgroup of ALL, point to a possible therapeutic use of autologous NK cells activated and expanded ex vivo. Management of Ph+ ALL patients - particularly the elderly - with TK inhibitors plus an immune-based strategy aimed at controlling/eradicating MRD without the use of systemic chemotherapy and/or transplant program is worthy of being investigated. Disclosures: No relevant conflicts of interest to declare.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.852 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A893-A893

Author(s):

Laurent Gauthier ◽

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.

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The TLR7/8 Ligand R848 Is Superior In Generation of Monocyte Derived Dendritic Cells From AML Patients for the Induction of Potent T and NK Cell Immune Responses

Blood ◽

10.1182/blood.v116.21.2195.2195 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2195-2195

Author(s):

Daniela Dorfel ◽

Barbara Beck ◽

Christiane Geiger ◽

Felix Lichtenegger ◽

Lysann Lindner ◽

...

Keyword(s):

Dendritic Cells ◽

Immune Responses ◽

Nk Cell ◽

Residual Disease ◽

Healthy Controls ◽

T Helper ◽

Tlr Agonists ◽

Migratory Capacity ◽

Healthy Donors ◽

Minimal Residual

Abstract Abstract 2195 Introduction: Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Dendritic cells derived from autologous peripheral blood monocytes have been tested as cellular adjuvants for therapeutic vaccination of malignancies and proven feasibility and safety, but overall clinical response rates remain very low. The vast majority of DCs used for clinical trials were differentiated with a standard maturation cocktail composed of the cytokines TNF-a, IL-1b, IL-6 and PGE2 leading to DCs unable to secrete biologically active IL-12. This cytokine is fervently desired because of its leading role in promoting T helper 1 cell polarization and therefore fostering the appropriate adaptive immune responses needed to combat minimal residual disease. Cocktails containing synthetic Toll-like receptors (TLR) agonists emerged as an attractive alternative for the induction of DC maturation with T helper type 1 polarizing capacity. Our present investigation was designed to study the feasability of a clinical grade DC 3-day mDC generation protocol from nonleukemic monocytes of intensively pretreated AML patients with novel maturation cocktails containing different TLR-agonists in vitro and assessment of their potency to induce adaptive and innate immune responses. Material & Methods: Monocytes isolated from peripheral blood of AML patients in CR and healthy donors were differentiated into immature DC with GM-CSF and IL-4. After 48 hours DC were additionally cultured with TNF-a, IL1-b, INF-g, PGE2 and corresponding to the defined cocktail with the TLR7/8 agonists R848 (R) or CL075 (C) with or without the TLR3 agonist poly(I:C) (P) for 24 hours. mDCs were analyzed for expression of maturation surface markers, costimulatory profile, IL-12(p70)/IL-10 ratio, migratory capacity, NK cell activation and polarization of T cells. Results: No significant difference in absolute monocyte counts and percentage of DC recovery between healthy controls and AML patients in CR was found using different maturation cocktails (C, CP, R, and RP). Phenotype analysis of surface marker expression revealed no substantial differences between the different DC generation protocols used in healthy donors and AML patients in CR. The costimulatory profile assesed by the expression of two members of the B7 family, CD80 (B7.1) and CD274 (B7-H1 or PD-L1), was in healthy donors superior to AML patients, but these differences were not statistically significant. Variations were noted in the capacity of DCs derived from different donors to produce IL-12(p70) and IL-10, but importantly no significant differences between AML patients in CR and healthy controls could be observed. Interestingly, both healthy donor and AML derived DCs secrete a significantly higher proportion of IL-12(p70) with R848 containing cocktails compared to CL075. Treatment with the CP cocktails even leads to a inverse Il12/IL-10 ratio in AML patients. The high CCR7 expression was paralleled by a strong migratory capacity as well as positive chemotactic reponses to CCL19 chemokine signals. DCs matured with these novel cocktails induced potent alloresponses and strongly activated NK cells measured by upregulation of CD69 expression and IFN-g secretion. No differences beetween R848 and CL075 could be observed. Conclusion: Here we report for the first time a clinically applicable, time- and resource saving 3-day TLR-agonist containing maturation protocol for the generation of IL-12(p70) secreting DCs from AML patients in remission validated with healthy controls which allowed efficient generation, easy harvesting, stable maturation and substantial recoveries of mature DCs. Comparison of different TLR7/8 agonists showed superiority of R848 in IL-12(p70) production to CL075. We believe that these studies point the way to improved DCs that will induce better and long lasting immune responses in the vaccination against acute myelo Disclosures: No relevant conflicts of interest to declare.

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Prevention Of Minimal Residual Disease In Ph+ ALL

Blood ◽

10.1182/blood.v122.21.1265.1265 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1265-1265

Author(s):

Iris Appelmann ◽

Elisa DeStanchina ◽

Gregory Carbonetti ◽

Chong Chen ◽

Scott W. Lowe ◽

...

Keyword(s):

B Cells ◽

Minimal Residual Disease ◽

Residual Disease ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Janus Kinase ◽

Response To Treatment ◽

Cytokine Signaling ◽

Minimal Residual

Abstract Aggressive Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease can be induced by the introduction of cultured p185 (BCR-ABL)-expressing Arf-null pre/pro-B cells into healthy, unconditioned syngeneic mice. Only 20 polyclonal donor cells are sufficient to induce lethal ALL within 30 days of their IV administration, indicating that BCR-ABL expression and Arf inactivation are sufficient to guarantee leukemogenesis in healthy recipient animals. Leukemic mice enter transient remission in response to treatment with potent second generation tyrosine kinase inhibitors (TKI) such as dasatinib (SprycelTM). However, like human patients with Ph+ ALL, the continuously treated animals ultimately relapse with the emergence of leukemic clones containing clinically relevant BCR-ABL mutations, the nature of which depends upon the intensity of TKI treatment. Premature withdrawal of dasatinib when animals are in remission results in re-emergence of leukemia; surprisingly, leukemic B cells recovered from these animals lack BCR-ABL mutations and remain sensitive to dasatinib ex vivo. Hence, minimal residual disease depends upon salutary signaling within the hematopoietic microenvironment. In agreement, the response to TKI therapy can be significantly improved by abrogating cytokine signaling through a knockdown of the common gamma chain of the cytokine receptor. Administration of the Janus kinase (JAK) inhibitor ruxolitinib (Jakafi™) mimics this response. Although ruxolitinib demonstrated no anti-leukemic activity of its own, the overall survival of leukemic mice inoculated with 200,000 p185+ Arf-/- pre/pro-B cells was significantly extended after administration of a targeted combination therapy of ruxolitinib and dasatinib in comparison with mice treated with dasatinib alone. Addition of dexamethasone further reduced the leukemic burden, prevented CNS relapse, and led to prolonged survival. This implicates prevention of minimal residual disease and relapse by a non-toxic combination of targeted treatments. These studies have provided a rationale for a Phase I/II clinical trial employing these agents, particularly in older patients who are ineligible for bone marrow transplantation or do not tolerate cytotoxic chemotherapy. Disclosures: Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

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

Blood ◽

10.1182/blood.v124.21.5294.5294 ◽

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 >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.

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41BBL-Based Activation and Expansion of Autologous Natural Killer Cells Results in Enhanced Activity Against Leukemia Including ALL

Blood ◽

10.1182/blood.v124.21.2293.2293 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2293-2293

Author(s):

Ekta Kapadia ◽

Elad Jacoby ◽

Mark Kohler ◽

Waleed Haso ◽

Christopher Daniel Chien ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Enhanced Activity

Abstract Childhood leukemia is the most common pediatric malignancy. There are now excellent cure rates for these patients, however outcomes remain poor for those with refractory disease and for those who relapse after standard salvage therapies, with a disease recurrence of approximately 50%. Therefore, development of novel cellular therapies is essential to treat these refractory patients. Natural Killer (NK) cells generated from an allograft contribute to improved disease free survival after Hematopoietic Stem Cell Transplantation for leukemia when there is a KIR mismatch. This effect appears to be particularly potent in the setting of Acute Myelogenous Leukemia (AML) with less benefit demonstrated in Acute Lymphoblastic Leukemia (ALL). Preclinical studies have also suggested that activation and expansion of resting NK cells can enhance NK cell cytotoxicity and eliminate the need for KIR mismatch due to up-regulation of activating receptors. We are currently testing this approach in the clinic following a fully matched allogeneic transplant platform for leukemia. Our aim is to explore whether 41BB ligand (41BBL) and recombinant IL-15 (rIL-15) mediated ex vivo expansion of autologous NK cells results in enhanced activity against AML and ALL. The activation/expansion process may allow for the use of autologous NK cell infusions, thus eliminating the need for allogeneic NK cell donors. To test this hypothesis, we ex vivo expanded and activated NK cells derived from C57BL/6J (B6) mice using artificial Antigen Presenting Cells (aAPCs) containing 41BBL and rIL-15 for 7-14 days. NK cells were co-cultured with murine AML cells (C1498) and murine ALL cells (E2A-PBX) – both on B6 background. Controls included YAC cells (murine T-cell lymphoma cell line sensitive to NK cell killing) as well as Phorbol Myristate Acetate (PMA)/ionomycin. All cells were co-cultured for 5 hours prior to functional assessment of NK cells via CD107a degranulation. NK cells cultured with 41BBL aAPCs and rIL-15 had a 30-fold expansion in numbers (Figure 1) and an increase in purity to approximately 95-98% (NK1.1+, CD3–) by Day 7. In the absence of cytokine or aAPCs, cultured NK cells underwent rapid apoptosis. Functionally, although resting NK cells (harvested prior to assessment) expressed CD107a when cultured with YAC cells and PMA, only minimal degranulation was observed in the presence of autologous AML cells or ALL cells. In contrast, activated and expanded autologous NK cells displayed enhanced activity against ALL, AML, as well as YAC cells, while only minimal levels of CD107a were seen in the absence of targets (Figure 2). In vivo experiments with a single injection of activated and expanded NK cells did not result in prolonged survival of mice bearing either AML or ALL. Assessment of adoptively transferred NK cells demonstrated very transient persistence (<2 days) with no in vivo expansion, suggesting that repeated injections may be necessary for leukemia eradication. Future murine experiments will investigate the effect repeated injections of activated/expanded NK cells and/or the administration of rIL-15 will have on survival and leukemia eradication. In addition, the ability to activate and expand NK cells in culture provides an opportunity for lentiviral-based transduction with chimeric antigen receptor (CAR) vectors. We are currently testing this with a murine CD19 CAR. These experiments suggest that autologous activated and expanded NK cells may serve as a viable cellular therapy for pediatric patients with refractory/relapsed leukemia. As demonstrated in these in vitro experiments, autologous activated/expanded NK cells still show increased targeting of mouse AML and ALL cell lines despite the lack of KIR mismatch. Thus, they may serve as a potential platform for leukemia therapy, including ALL, which appear to be poor targets for resting NK cells. In addition, these cells demonstrate transient persistence in vivo, a potential advantage in the context of redirected cytotoxicity using CAR constructs that target antigens with broader expression in the hematopoietic compartment. Figure 1: <![if !vml]><![endif]> Figure 1:. <![if !vml]><![endif]> Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v126.23.3793.3793 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3793-3793 ◽

Cited By ~ 3

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.

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

Blood ◽

10.1182/blood-2021-149693 ◽

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 < 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 (<30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p<0.01, Figure C), blood (6-fold decrease, p<0.01), and liver (4-fold decrease, p<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<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<0.05), and IFN-γ production was reduced (p<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.

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Interleukin-15 Upregulates the Expression of NK-Cell Activating Receptors and Concomitantly Increases the NK Cytotoxicity in Patients with Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.2298.2298 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2298-2298

Author(s):

Miroslaw J. Szczepanski ◽

Malgorzata Czystowska ◽

Marta Szajnik ◽

Ann Welsh ◽

Kenneth A. Foon ◽

...

Keyword(s):

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Receptor Expression ◽

Activating Receptors ◽

Healthy Donors ◽

Interleukin 15 ◽

Nk Cell Cytotoxicity ◽

Nk Cytotoxicity ◽

Acute Myeloid

Abstract Natural killer (NK) cells lyse malignant cells without prior antigen-specific priming and play a critical role in the innate immune response. A balance of signals from activating and inhibiting receptors expressed on each NK cell controls its activity. The growth, differentiation and survival of NK cells have been found to be dependent on interleukin-15 (IL-15). Using multicolor flow cytometry we investigated the receptor repertoire and also measured the NK cell activity in twenty three patients with newly diagnosed acute myeloid leukemia (AML) prior to any treatment. Further, we investigated the ex-vivo effect of IL-15 on the NK cell repertoire and NK cell cytotoxicity. The percentage of circulating NK cells was lower (p<0.0001) in the AML patients (6%± 0.7, range 1–17%) compared to the NK cells of healthy donors (12%± 1, range 9–17%). The expression of the activating natural cytotoxicity (NCR) receptors NKp30 and NKp46 and the C-type lectin receptors NKG2D and NKG2C was significantly decreased in the AML patients compared to the NK cells of healthy donors: NKp30 24 vs 51% p<0.0001, NKp46 32 vs 73% p<0.0001, NKG2D 43 vs 83% p<0.0001, NKG2C 17 vs 28% p<0.03. In addition, the receptor expression (mean fluorescence intensity, MFI) was also significantly lower in AML patients compared to healthy donors. No significant differences in the expression of the NCR NKp44 and the NK- cell inhibitory receptors were observed. Furthermore, the NK cytotoxicity in the AML patients at diagnosis was significantly lower (p<0.0003) compared to the NK cytotoxicity of healthy donors (4 vs 75 LU). When NK cells obtained from AML patients were cultured with IL-15, significant increases in the expression of the NK- cell activating receptors (Table 1) were observed. The upregulation of the activating receptors was associated with a concomitant significant increase (p<0.001) of the NK cell cytotoxicity (4 vs 70 LU). The data suggest that IL-15, a homeostatic NK cell cytokine, can upregulate the expression of activating receptors and concomitantly increase the NK lytic activity. The use of IL-15 as a platform for NK- based therapies for AML patients should be considered in the future. Table 1. The effect of IL-15 on the receptor expression in AML patients NK cell Activating Receptors NK cells in AML pts at diagnosis NK cells in AML pts after IL-15 stimulation p value % positive, MFI % positive, MFI NKp30 24, 2.6 84, 6.0 0.001 NKp44 4, 2.4 71, 6.7 0.001 NKp46 32, 2.6 83, 7 0.002 NKG2C 17, 2.2 58, 6.9 0.005 NKG2D 43, 2.5 87, 7.8 0.01

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Artificial Antigen Presenting Cells (aAPC) Expanded NK-Mediated Enhanced Lysis of Allogeneic Tumor Cells Regardless of HLA Class I/KIR Mismatch and Its Implication of Use in Eradicating Acute Lymphoblastic Leukemia (ALL).

Blood ◽

10.1182/blood.v114.22.3023.3023 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3023-3023

Author(s):

Hua Zhang ◽

Bruce Levine ◽

Nga Voong ◽

Alan S. Wayne ◽

Carl H. June ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Tumor Cells ◽

Fusion Proteins ◽

Nk Cell ◽

Lymphoblastic Leukemia ◽

Target Cells ◽

Activating Receptors

Abstract Abstract 3023 Poster Board II-999 NK Killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands play critical roles in maintaining natural killer (NK) cell tolerance, while providing surveillance against pathogens and malignant transformation. Natural killer (NK) cells have been explored as tools for adoptive anti-tumor or leukemia immunotherapy and current models hold that a mismatch or absence of KIR ligands on target cells is essential for efficient NK cell mediated cytolysis. However, new approaches are now available to activate NK cells and the role for KIR mediated signaling in regulating cytotoxicity of activated NK cells has not been well studied. In this study, aAPCs comprising IL15Ra+K562 cells engineered to express 4-1BBL activated and expanded peripheral NK cells in the presence of exogenous IL15 up to 1000-fold in 3 weeks. Compared to resting NK cells, 4-1BBL/IL15-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and showed significantly enhanced cytotoxicity against a multitude of tumor targets including K562, Daudi, Ewing's tumors, osteosarcoma, as well as autologous tumors (50%-90% killing vs. 0%-8% with non-activated NK cells). Meanwhile we could detect little to no influence of KIR signaling in regulating cytotoxicity by aAPC activated NK cells, since sorted CD158a+ and CD158b+ activated NK cells showed similar killing of tumor cells expressing HLA group C1 (CD158b ligand) and/or C2 (CD158a ligand) antigens. In contrast, killer activating receptors (KARs) were indispensable for the cytolysis of solid pediatric tumors by aAPC-activated NK cells, since the killing was significantly inhibited by fusion proteins binding to the ligands of NKG2D, NK p30, p44, p46, p80 (KARs). About 20-40% inhibition of the killing was accomplished when all four activating receptors were blocked, though other activating receptors have not been well defined. Although acute lymphoblastic leukemia (ALL) blasts were refractory to fresh NK cytotoxicity, 4-1BBL/IL15 activated NK cells demonstrated higher lytic activities (20%-50%) against ALL blasts from either patients or cell lines. ALL blast lysis could be completely or partially inhibited by KAR-blocking fusion proteins, indicating that expression levels of KAR ligands vary among ALL cases and other solid tumors. We conclude that KIR ligand mismatch or absence is not essential for effective NK cytotoxicities on either solid tumors or ALL when fully activated NK cells are utilized. This suggests that adoptive therapy with autologous aAPC-activated NK cells may prove effective in some clinical settings, such as ALL, AML, or certain solid tumors. Further studies to assess the impact of KAR ligand expression on aAPC-activated NK killing of ALL blasts are in progress. Percentage of Activated NK Killings vs. Fresh NK's with/without KAR-Ig Fusion Proteins Activated NK (E:T=2.5:1) Fresh NK (E:T=25:1) -KAR-Ig Fc +KAR-Ig Fc SB tumor (Ewing's) 48% 30% 0.5% HOS (Osteo sarcoma) 63% 36% 0.7% Daudi (B. lymphoma) 78% 46% 0.2% REH (ALL) 54% 8% 3% Disclosures No relevant conflicts of interest to declare.

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Patterns of Individual Tumor Response Testing (ITRT) and Bone Marrow Minimal Residual Disease (MRD) On Day 15 of Therapy in Childhood Precursor-B-Lineage Acute Lymphoblastic Leukemia (ALL): MRD and Ex Vivo Drug Resistance Have Similar Predictive Value of Relapse.

Blood ◽

10.1182/blood.v120.21.2448.2448 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2448-2448

Author(s):

Jan Styczynski ◽

Anna Jaworska-Posadzy ◽

Malgorzata Kubicka ◽

Robert Debski ◽

Beata Kurylo-Rafinska ◽

...

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Multivariate Analysis ◽

Residual Disease ◽

Ex Vivo ◽

Cox Model ◽

Lymphoblastic Leukemia ◽

Minimal Residual ◽

Risk Of Relapse

Abstract Abstract 2448 Introduction: The speed of blast clearance during therapy is a major prognostic factor of outcome in childhood acute lymphoblastic leukemia (ALL). Blast count in the peripheral blood on day 8, or in the bone marrow on day 15 and day 33, have been widely used to deliver risk-directed therapy. Another approach to measure the speed of leukemia clearance is the detection of minimal residual disease during induction therapy, as well as at days 33 and 78 of therapy. In vitro measurements of drug resistance (called recently as ITRT, individual tumor resistance testing) in leukemic cells obtained at diagnosis have been of prognostic significance in the prediction of clinical outcome in selected groups of patients. Objective: The analysis of the prognostic impact of (A) residual disease (MRD) at day 15 of induction therapy; (B) in vitro drug resistance at diagnosis (ITRT), (C) correlation of MRD and ITRT, and (D) multivariate analysis of prognostic role of MRD, ITRT, initial factors and initial therapy response to the risk of relapse. Patients and Methods: A total number of 87 children (aged 1–18 years) diagnosed for pre-B-ALL, treated either with ALL-BMF-90 or ALL-IC-2002 protocol were included into the study. ITRT was tested at diagnosis by the MTT assay. Residual disease at day 15 was measured by flow cytometry and determined for cut-off value BML15<0.5%. The median follow-up was 8.9 yrs (range, 0–11.5). Following drugs were tested: prednisolone, dexamethasone, vincristine, L-asparaginase, daunorubicin, doxorubicin, etoposide and cytarabine. PVA score was determined as combined ITRT profile to prednisolone, vincristine and L-asparaginase. Results: (A) The overall pDFS was 0.721±0.052 and the mean survival 9.1 yrs (95%CI=8.2–9.9). Patients with BML15<0.5% had pDFS=0.816±0.055, while those with BML15>0.5% had pDFS=0.542±0.098 (p=0.009, log-rank). The risk of relapse in BML15-positive patients was 3.0-fold higher (1.3–7.1, p=0.013). (B) pDFS was significantly better for patients with sensitive ITRT profile to: PVA (1.00±0.00 vs 0.61±0.06, p=0.002), prednisolone (0.89±0.05 vs 0.54±0.08, p=00002), vincristine (0.84±0.06 vs 0.61±0.08, p=0.035), daunorubicin (0.094±0.04 vs 0.51±0.08, p=0.00002), and L-asparaginase (0.84±0.06 vs 0.59±0.08, p=0.009). In multivariate analysis in Cox model, the prognostic value was retained only for ITRT for prednisolone (p=0.013, HR=0.08, 95%CI=0.01–0.6) and daunorubicin (p=0.004, HR=0.05, 95%CI=0.01–0.4), while ITRT for PVA score was below of significance (p=0.068, HR=0.03, 95%CI=0.01–1.3). (C) Patients with MRD-positive ALL at day 15 (BML15>0.5%) had higher ITRT for following drugs: doxorubicin (p=0.005, RR=1.8, Mann-Whitney U test), L-asparaginase (p=0.029, RR=3.2), and etoposide (p=0.055, RR=4.1), while no differences were found for other drugs. In multivariate logistic regression, the significance impact to development of BML15>0.5% was found for doxorubicin (p=0.035, OR=0.33) and etoposide (p=0.048, OR=0.14). (D) In multivariate analysis in Cox model for relapse risk, three factors had predictive value: BML15>0.5% (p=0.010, OR=3.3, 95%CI=1.3–8.2), ITRT for prednisolone (p=0.012, OR=4.4, 95%CI=1.4–13) and ITRT for daunorubicin (p=0.018, OR=5.9, 95%CI=1.4–26), while age, prednisolone-poor-response at day 8, BM response at day 15, BM response at day 33, and BCR-ABL rearrangement had no significant value. Conclusions: Patients with residual disease at day 15 had 3-fold higher risk of relapse. Patients with resistant ITRT profile to prednisolone and daunorubicin had respectively 12- and 20-fold higher risk of relapse. Presence of residual blasts at day 15 correlates with ITRT to etoposide and doxorubicin. Finally, persistence of blast in marrow at day 15 (BML15>0.5%) and ex vivo drug resistance (ITRT) to prednisolone and to daunorubicin were the strongest prognostic factors predicting relapse in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

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