scholarly journals KIR Gene Haplotype: An Independent Predictor of Clinical Outcome in MDS Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4330-4330
Author(s):  
May Daher ◽  
David Marin ◽  
Catherine Sobieski ◽  
Hila Shaim ◽  
Rafet Basar ◽  
...  
Keyword(s):  
Nk Cells ◽  
Disease Progression ◽  
Research Funding ◽  
Nk Cell ◽  
Risk Group ◽  
Clonal Evolution ◽  
Median Number ◽  
Gene Repertoire ◽  
Chromium Release Assay ◽  
Haplotype A

Abstract Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders affecting the myeloid lineage, characterized by cytopenias and clonal evolution to AML. Immune responses against MDS, partly mediated by NK cells, have the potential to affect disease progression. However, immune evasion remains an important barrier. NK cells express both activating and inhibitory killer immunoglobulin-like receptors (KIRs) that interact to regulate NK effector function. Based on the number and distribution of inherited KIR genes, individuals can be classified in two broad haplotypes. Haplotype A comprises a single activating KIR (aKIR) gene (KIR2DS4), while haplotype B incorporates various combinations of aKIR genes (up to 6). We hypothesized that the aKIR gene repertoire may be useful in refining predictions of clinical outcomes in MDS. Thus, we studied the variations in aKIR gene content and haplotype in MDS and their relationship to the risk of AML transformation and patient outcomes. We first compared the number of aKIR genes in 108 MDS patients treated at MDACC with that in 139 HSC donors. The median number of aKIR genes was significantly lower in MDS than controls: 2 (range 0-6) vs 3 (range 0-6), p=0.001. Functional studies revealed that compared to healthy controls, NK cells from MDS patients demonstrate less Interferon gamma production (p<0.0001) and less degranulation (p=0.0028) in response to K562 cell line, and have lower killing ability evidenced by chromium release assay (p=0.04). We next examined the influence of KIR haplotype on the risk of AML transformation and outcomes in the cohort of 108 MDS patients (28 KIR haplotype A and 80 KIR haplotype B patients). On multivariate analysis, cytogenetic risk group and KIR haplotype were identified as independent predictors of MDS progression to AML. The relative risk of MDS-AML transformation in patients with haplotype A vs. haplotype B was 2.67 (1.13-6.31) (p=0.02). Similarly, cytogenetic risk group, IPSS and KIR haplotype independently predicted survival. MDS patients with KIR haplotype A had worse adjusted PFS (RR with 95%CI 2.96 [1.59-5.52], p=0.001) and OS (2.25 [1.17-4.31], p=0.02), compared to patients with haplotype B. These novel findings may help identify a subgroup of MDS patients with a high risk of disease progression and poor outcomes, who would likely benefit from adoptive NK cell therapy. Disclosures Kantarjian: ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

scholarly journals Clinical Significance of KIR Genotype in Patients with Methotrexate-Associated Lymphoproliferative Disorders

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5219-5219
Author(s):  
Norina Tanaka ◽  
Aya Watanabe ◽  
Suguru Honda ◽  
Mitsuko Kobayashi ◽  
Yan-Hua Wang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Function ◽  
Nk Cell ◽  
Hla Class I ◽  
Lymphoproliferative Disorders ◽  
Advisory Committees ◽  
Kir Genes ◽  
Haplotype A

Background: Methotrexate-associated lymphoproliferative disorders (MTX-LPD) develops in patients with rheumatoid arthritis (RA) or other autoimmune disorders during low-dose MTX treatment. MTX-LPD includes wide variety disease spectrum, ranging from polymorphic proliferation to aggressive lymphoma. Although etiology of MTX-LPD has not been fully understood, approximately half of the MTX-LPD cases showed association with EB virus (EBV), suggesting that MTX treatment causes reduced immune response to EBV-positive cells, and results in MTX-LPD development. Natural killer (NK) cells play important roles in eradicating tumor and virus-infected cells. NK cell function is modulated by multiple cell surface receptors, including Killer immunoglobulin-like receptor (KIR). There are multiple KIR genes (inhibitory or activated), which are various in number and/or composition among individuals, on chromosome 19q. Previous reports demonstrated that combination of KIR genes affects NK cell function, and is associated with the risk of development of certain types of cancers, viral infections and collagen disease. There is no report about the association of KIR genotype and MTX-LPD. We consider that NK cells play a significant role in suppression of MTX-LPD development. In this study, we focused on examining genotype KIR and KIR-ligand (HLA class I). Methods: We retrospectively analyzed 35 MTX-LPD cases diagnosed between 2009 and 2019. Genomic DNA was extracted from mononuclear cells that were isolated from the bone marrow or peripheral blood samples of patients with MTX-LPD. KIR genotypes were analyzed using the KIR genotyping sequence-specific primers kit. The variations of KIR content and haplotype and their relationship with progression to malignant lymphoma (ML) and response to chemotherapy were investigated. HLA was analyzed using PCR-Luminex assay. The frequency of each HLA allele and each combination was determined by referring to the data base of an HLA laboratory. Chi-squared (χ2) tests and Wilcoxon rank sum tests were used to test associations between the variables. Results: Among the 35 patients, 25 were diagnosed with ML and 10 with polymorphic LPD. Diffuse large B cell lymphoma (DLBCL) was most common type in ML (57.7%). Table 1 showed characteristics of patients and summary of the results. All patients underwent MTX treatment for RA. The median duration of MTX administration at the time of MTX-LPD diagnosis is 11.5 years (range=0.8-27.2), and median MTX dose was 10mg/week (range=4-17.6). The duration and dose of MTX had no effect between ML and polymorphic LPD. Twenty-three patients required chemotherapy, and 12 patients had tumor regression after stopping MTX treatment. Relative patient populations requiring chemotherapy in ML or polymorphic LPD were 85% or 11%, respectively (P=0.0001). EBV-positive patients tended to regress tumors with MTX discontinuation alone (P=0.16). In KIR genotype analysis, patterns of number and combination of the KIR genes are mainly classified as haplotype "A" containing multiple inhibitory KIR genes with a KIR 2DS4 (an activated KIR [aKIR]) and haplotype "B" (other than haplotype "A"). Patients were classified in haplotype A (13 cases, 37%) and haplotype B (22 cases, 63%), respectively. ML patients showed higher ratio in haplotype A (ML 46.2% vs LPD 11.1% P=0.045). There was no difference in number of aKIR or iKIR between ML and polymorphic LPD patients. In HLA Class I analyses, there was significant difference in frequencies of HLA-C haplotype between lymphoma and polymorphic LPD patients (P=0.026). Furthermore, HLA- C1 / C1 patients were more relapsed or refractory to chemotherapy than C1 / C2 patients (P = 0.17). Conclusion: This is the first report showing clinical significance of KIR genotypes in MTX-LPD. Patients with haplotype A, a suppressive haplotype, seems to be at high risk for developing lymphomas that require chemotherapy during MTX treatment. HLA- C1/C1 patients are more likely to develop lymphomas that respond poorly to treatment, suggesting that the activity of NK cells may be lower because ligands can match with KIRs that are more restricted than C1/C2. Considering the potential NK functions with KIR genotype would improve the understanding of the prognosis and lead to prevention for MTX-LPD. Disclosures Hagiwara: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Harigai:Bristol Myers Squibb Co: Other: personal fees, Research Funding; Eisai Co: Other: personal fees, Research Funding; Ayumi Pharmaceutical Co: Other: personal fees, Research Funding; AbbVie Japan GK,: Other: personal fees, Research Funding; Eli Lilly Japan K.K: Other: personal fees; Kissei Pharmaceutical Co.: Other: personal fees; Teijin Pharma Ltd: Other: personal fees, Research Funding; Mitsubishi Tanabe Pharma Co: Research Funding; Nippon Kayaku Co.: Research Funding; Pfizer Japan Inc.: Other: personal fees; Chugai Pharmaceutical Co., Ltd.: Other: personal fees; Japan College of Rheumatology: Other: personal fees; Boehringer Ingelheim Japan, Inc: Other: personal fees; GlaxoSmithKline K.K: Other: personal fees; Oxford Immuotec,: Other: personal fees. Tanaka:Bristol-Myers Squibb: Research Funding.

scholarly journals Single-Cell Transcriptomic Analysis Reveals Loss of Activated Bone Marrow NK Cells in Multiple Myeloma Patients Which Associates with Disease Progression in Mice

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1578-1578
Author(s):  
Sabrin Tahri ◽  
Zoltan Kellermayer ◽  
Madelon M.E. de Jong ◽  
Natalie Papazian ◽  
Cathelijne Fokkema ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Single Cell ◽  
Disease Progression ◽  
Research Funding ◽  
Nk Cell ◽  
Newly Diagnosed ◽  
Cell Compartment ◽  
Tumor Load

Abstract Introduction Multiple Myeloma (MM) disease progression and therapy response are the net result of tumor cell-intrinsic features and tumor cell-extrinsic cues from the bone marrow (BM) microenvironment. Natural killer (NK) cells are mediators of the cytotoxic immune response against MM and are important effector cells in antibody-based immune therapies, especially anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided into a cytotoxic CD56 dim subset, important for antibody-dependent cellular cytotoxicity, and a cytokine-producing CD56 bright subset releasing inflammatory mediators such as IFNγ, TNFα and GM-CSF. However, accumulating evidence suggests greater heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact disease progression and response to NK cell-driven immunotherapies. Here, we combined the 5TGM1 murine model of MM with single-cell RNA sequencing of bone marrow (BM) NK cells of newly diagnosed MM patients to map NK cell heterogeneity and to investigate their role in MM progression. Results To gain insight in NK cell heterogeneity in MM disease we performed single-cell RNA sequencing on immune cells of viably frozen BM aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. Conventional CD56 bright and CD56 dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK +CD56 bright NK cells contained clusters of naïve and activated NK cells. The GZMB+CD56 dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets, we compared GZMK +CD56 bright vs GZMB+CD56 dim cluster composition and distribution between controls and MM patients. Control BM was dominated by GZMB-transcribing cytotoxic CD56 dim NK cells, resulting in a low ratio of cytokine-producing GZMK +CD56 bright vs cytotoxic GZMB+CD56 dim NK cells. In contrast, MM bone marrow was characterized by heterogeneity of this ratio with a subset of patients presenting with complete reversal of this ratio . In this subset of patients, the altered composition was due to a loss of cytotoxic GZMB +CD56 dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation. To better examine the significance of cytotoxic NK cells in MM disease course we utilized the well-established 5TGM1 mouse model. C57Bl/6 and KaLwRij mice both received 10 6 5TGM1-GFP cells intravenously. Three weeks after tumor injection all KaLwRij mice (18/18) developed MM, defined by &gt;5% tumor cells in BM ("unrestrained tumor") and serum M-protein &gt;2mg/ml. Interestingly, while 39% (7/18) of C57Bl/6 mice had no tumor, 44% (8/18) had low but detectable levels of MM cells (0.1-5% of BM cells, "restrained tumor") and 17% (3/18) presented with an unrestrained MM with BM tumor load similar to that seen in KaLwRij mice. With time the percentage of mice with unrestrained tumor increased (5/12, 42%) at the expense of restrained tumor (2/12, 16%). We hypothesized that C57Bl/6 mice with low tumor load could represent a model of immune-mediated tumor control. Detailed analysis of the NK cell compartment revealed an expansion of activated mature (CD69 + CD11b +CD27 +) NK cells in C57Bl/6 mice with restrained BM MM (p=0.0031). In contrast, high BM tumor burden in both genotypes was associated with a sharp decline in absolute numbers of activated NK cells. Conclusion: Through a combination of single-cell transcriptomic analyses of the BM immune microenvironment in MM patients and experimental mouse models we found a loss of activated NK cells in a subset of patients and mice. Our data suggests that loss of these activated NK cells is associated with MM progression in vivo. A subset of MM patients presented with a loss of activated cytotoxic GZMB +CD56 dim NK cells in the BM, suggestive of reduced cytotoxic anti-tumor responses. Meanwhile, in vivo, high disease burden only occurred in mice with an absence of activated NK cells. Current analyses are focused on differences in human disease progression and efficacy of Daratumumab-based therapies in patients with various NK cell phenotypes. Disclosures Broijl: Janssen, Amgen, Sanofi, Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

scholarly journals Haploidentical Mbil-21 Ex Vivo Expanded NK Cells (FC21-NK) for Patients with Multiple Relapsed and Refractory Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Stefan O. Ciurea ◽  
Jolie Schafer ◽  
Piyanuch Kongtim ◽  
Julianne Chen ◽  
Doris Soebbing ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Median Number ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Nk Cell Therapy

Background: Allogeneic stem-cell transplantation (alloSCT) remains the only curative treatment for patients with advanced AML. However, only a minority of these patients achieve disease control prior to transplantation. Natural Killer (NK) cells have potent anti-leukemic activity but are functionally deficient in AML. Adoptive NK-cell therapy using high-doses of functionally active NK-cells could overcome these limitations. We previously developed an ex vivo NK-cell expansion method based on K562 feeder cells modified to express membrane bound IL-21 (mbIL-21) and 4-1BB ligand, (FC21), which resulted in high numbers of hyperfunctional FC21-NK cells with enhanced cytotoxicity and cytokine production. Here we report outcomes of a phase I clinical trial designed to assess the safety, feasibility and maximum tolerated dose (MTD) of haploidentical FC21-NK cells for patients with relapse/refractory (R/R) AML at MD Anderson Cancer Center. Methods: Eligible patients were ≥18 years, KPS ≥70 with good organ function. Patients with relapsed AML after alloSCT were eligible if they had no active GVHD and did not require immunosuppression. Haploidentical donors were selected based on KIR characteristics, when multiple donors were available. Donor NK cells were expanded over 3 weeks and cryopreserved. Three dose levels between 106-108 cells/kg were planned. Patients received cytoreductive chemotherapy with fludarabine 30 mg/m2/day and cytarabine 2 g/m2/day for 5 days (4 days for age &gt;60) and G-CSF (subsequently eliminated). 3-7 days after chemotherapy, patients received FC21-NK cell infusions 3 times per week, up to 6 infusions. Results: As of 4/14/2020, 15 patients were screened, 12 of whom were eligible and received the FC21-NK cells. Median age was 60 years (range 25-70); 6 (50%) had adverse cytogenetics, 8 (66.7%) had adverse ELN genetic risk, 6 (50%) had primary induction failure, 2 (16.7%) had CNS disease and 4 (33.3%) had secondary AML. Median number of prior treatment regimens was 5 (range 2-8), median blast count at enrollment was 47% (range 7-88). Median time from diagnosis to enrollment and to first NK-cell infusion was 16.6 (range 2.5-98.1) and 17.2 (range 3.1-98.6) months, respectively. Donor-recipient NK-cell alloreactivity was seen in 5 patients (41.7%). Median number of NK-cell infusion was 6 (range 3-6); 8 (66.7%) and 4 (33.3%) patients received NK-cell dose of 1 X106 and 1 X107 cells/kg, respectively. MTD was not reached. Seven patients had ANC recovery post-NK cell infusion with cumulative incidence (CI) of ANC recovery to 500/mm3 at 60 days of 58.3%. Eight patients (66.7%) achieved complete remission (CR) (N=4, 33.3%) or CR with incomplete hematologic recovery (CRi) (N=4, 33.3%) at 30 days post-NK cell infusion. One patient with CR had negative minimal residual disease (MRD). Five patients (41.7%) proceeded to haploidentical alloSCT from the same donor and were transplanted in CR/CRi, all but one with persistent MRD. With a median follow-up of 13 months (range 4.1-42.7), median OS and DFS were 17.6 and 3.3 months, and 28 and 20 months for patients receiving alloSCT, respectively. Other outcomes including 2-year OS, DFS, relapse and TRM are shown in Figure 1 and Table 1. No infusion related toxicity or cytokine release syndrome was observed. Two patients were evaluable for FC21-NK cell persistence with haplotype-specific anti-HLA antibodies. FC21-NK cells were detected 5 and 6 weeks after the last FC21-NK cell infusion, respectively. A progressive decrease of the blast population with progressive expansion of the FC21-NK cell population after repeated NK-cell infusions was noted in samples collected from one pt (Figure 2). Persistence is also being evaluated by STR chimerism. Conclusions: Multiple infusions of FC21-NK cells yielded unprecedented outcomes with 66.7% of patients responding and approximately half proceeding to alloSCT in a heavily pre-treated, ultra-refractory, high-risk patient population. Responses were observed irrespective of dose. FC21-NK cell therapy was very well tolerated with no attributable AEs and were shown to persist for at least 5 weeks after infusion. These encouraging results warrant further clinical evaluation of FC21-NK cells in R/R AML patients. Disclosures Ciurea: Kiadis Pharma: Current equity holder in publicly-traded company, Research Funding. Schafer:Kiadis Pharma: Current Employment. Shpall:Zelluna: Membership on an entity's Board of Directors or advisory committees; Adaptimmune: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Licensing Agreement. Konopleva:Calithera: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Forty-Seven: Consultancy, Research Funding; Sanofi: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Ascentage: Research Funding; Rafael Pharmaceutical: Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Omeros: Consultancy; DKMS America: Membership on an entity's Board of Directors or advisory committees; Cytonus: Consultancy; Genzyme: Speakers Bureau; Takeda: Patents & Royalties.

scholarly journals BMT CTN 1803: Haploidentical Natural Killer Cells (CSTD002) to Prevent Post-Transplant Relapse in AML and MDS (NK-REALM)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1955-1955
Author(s):  
Sumithira Vasu ◽  
Nelli Bejanyan ◽  
Steven Devine ◽  
Elizabeth Krakow ◽  
Elizabeth Krakow ◽  
...  
Keyword(s):  
High Risk ◽  
Nk Cells ◽  
Cumulative Incidence ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Marrow Transplant ◽  
Free Survival ◽  
Blood And Marrow Transplant ◽  
Relapse Rates

Background and Rationale: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic blood or marrow transplantation (BMT). Although relapse rates vary based on patient population, age, and conditioning intensity, relapse is experienced in at least 30-50% after conventional BMT in high-risk AML/MDS. Initial safety and post-BMT relapse risk reduction results are reported by investigators at MD Anderson Cancer Center in a phase I study of ex vivo-expanded, donor-derived, haploidentical natural killer (NK)-cell infusion in conjunction with haploBMT. Of 13 patients with high-risk myeloid malignancies treated with NK cells, no infusion reactions or dose-limiting toxicities occurred and only 1 patient, treated at the lowest dose of 1×105 cells/kg, relapsed (Ciurea, Blood 2017). This experience supports investigation of CSTD002, a product derived from haploidentical donor NK cells and expanded ex vivo using plasma membrane (PM21) nanoparticles bearing membrane-bound IL-21 and 4-1BBL. This study represents a public-private partnership between the sponsor (Kiadis Pharma) and the Blood and Marrow Transplant Clinical Trials Network (BMT CTN), leveraging existing National Institutes of Health-supported clinical trials infrastructure to conduct a complex cellular immunotherapy trial. We used contemporary, unpublished data from the Center for International Blood and Marrow Transplant Research registry to determine baseline relapse rates that informed the statistical design. Doses of NK cells expanded by a novel method and exceeding those previously achieved in most published studies will be given in the peri-transplant period to test the hypothesis that haploidentical NK cells can mediate an effective anti-leukemia response. Trial Design and Methods: BMT CTN 1803 is a phase II, single-arm, open-label, multicenter trial designed to investigate the safety and efficacy of CSTD002 for the treatment of patients with high-risk AML or MDS undergoing haploBMT. An initial safety run-in phase will precede enrollment into the full study of approximately 60 patients. Major inclusion criteria of patients and donors are listed in the Table. Peripheral blood will be drawn from the donor to start the NK-cell expansion approximately 5 weeks before the planned haploBMT. Patients will receive intravenous (IV) melphalan 140 mg/m2 (100 mg/m2 for patients ≥60 years old) on Day -7; fludarabine 40 mg/m2 IV on Days -7, -6, -5, and -4; and 2 Gy of total body irradiation on Day -3. Donor bone marrow will be harvested and given on Day 0. Three doses of CSTD002 will be administered IV on Days -2, +7, and +28, relative to the haploBMT. The recommended dose of CSTD002 for administration will be formulated at 1×108 NK cells/kg of recipient body weight. Graft-versus-host disease (GVHD) prophylaxis is post-transplantation cyclophosphamide with tacrolimus and mycophenolate mofetil. The primary endpoint is cumulative incidence of relapse at 1 year post haploBMT in patients receiving at least 1 infusion of CSTD002. Secondary endpoints are safety and tolerability of CSTD002; overall survival; non-relapse mortality; relapse-free survival; GVHD-free survival; cumulative incidence of acute GVHD and chronic GVHD; hematologic recovery; donor-cell engraftment; primary and secondary graft failure; overall incidence of toxicity; and cumulative incidence of infections including cytomegalovirus re-activation and symptomatic BK virus hemorrhagic cystitis. Exploratory endpoints are systemic immunosuppression-free survival; immune reconstitution at Days 28, 100, and 365 post haploBMT; proportion of patients with detectable minimal residual disease at Days 28 and 100 post haploBMT; feasibility of administering the planned CSTD002 doses; and impact of NK-cell alloreactivity on relapse and survival. Disclosures Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: Clinical trial support. Bejanyan:Kiadis Pharma: Other: advisory board. Devine:Kiadis Pharma: Other: Protocol development (via institution); Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta; Bristol Myers: Other: Grant for monitoring support & travel support. Krakow:Bellicum Pharmaceuticals: Research Funding; Highpass Bio: Research Funding; Magnolia Innovations: Other: Personal fees. Logan:Eisai: Other: Personal fees; Astellas: Other: Grant; Kiadis (formerly Cytosen): Other: Grant; Novartis: Other: Personal fees; Kite: Other: Grant. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder. Barrett:Kiadis Pharma (formerly Cytosen): Other: Personal fees; Biologics Consulting Company: Other: Personal fees. Shan:Kiadis Pharma (formerly Cytosen): Employment. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Sanofi-Genzyme: Research Funding.

NK Cytotoxicity Is Decreased During the Early Posttransplant Period Following Pediatric Allogeneic Transplantation From Unrelated Donors Using CD3/CD19-Depleted Graft.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2434-2434
Author(s):  
Antonio Pérez-Martínez ◽  
Manuel Ramírez ◽  
María Ruiz-Salmerón ◽  
Marta Gonzalez-Vicent ◽  
S. Grande ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Nk Cell ◽  
Cell Subset ◽  
Median Number ◽  
Hematopoietic Stem ◽  
Unrelated Donors ◽  
Nk Cytotoxicity

Abstract Abstract 2434 Poster Board II-411 Introduction and objectives: Unrelated donors, match unrelated (MUD) and haploidentical donors (HSCT), have been described as a therapeutic option for high-risk childhood acute leukemia. CD3/CD19 depleted graft has been used in order to decrease the incidence of graft versus host disease (GvHD) and post-transplant lymphoproliferative disease, in the unrelated transplantation setting. Donor-derived NK cell alloreactivity has been reported to mediate early graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation. NK cells are components of the innate immunity playing an important role in the surveillance of human tumors. NK cell recognition of malignant cells depends on a dynamic balance between activating and inhibitory receptors. NK cell alloreactivity can be predicted by donor Killer Immunoglobulin like Receptors (KIRs), Natural Killer Receptors (NCRs), C-type Lectin receptors (NKG2D), Toll Like Receptors (TLRs) and recipient human leukocyte antigen (HLA) class I alleles as ligands. Reduced risk of relapsed has been described in malignant cancer after haploidentical stem cell transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient (KIR–HLA receptor–ligand mismatch). We prospectively investigated NK function and NK reconstitution in 18 CD3/CD19 depleted graft unrelated hematopoietic stem cell transplantation (7 MUD and 11 HSCT) using fludarabine-based reduced intensity conditioning regimen. Results: NK cells peaked around day 30 after transplantation. The median number of NK cells on day +30 was 403±88/μL . On day 100 after transplantation the median number of NK cells/μL was 221±58. While the CD56bright NK cell subset was above normal during the first 100 days post-transplant, the “effector” NK cell subset, CD56dim CD16bright, was significantly reduced early after transplantation. The median percentage of CD56bright cells among NK cells in peripheral blood was 25.8±4.6% at day +30, and it was 24.5±5.7 at day +100. The decreased in CD56dim CD16bright NK cell subset was correlated with the decreased of the inhibitory KIR receptors (KIR2DL1, KIR2DL2, KIR3DL1) expression. We also observed a lower expression than donors of the activating receptors NKG2D, TLR4 at day +30, NKp46, TLR 9 at day 60 and NKp46, NKp30 at day +100. Although absolute NK-cell counts rapidly increased after transplant, their cytotoxicity against K562 was much lower compared to that of their donors. At day 100 after transplantation, patients NK cytotoxicity was lower than donor values. These results suggest that the low NK cell cytotoxicity could be related to an “immature” NK phenotype during the early period after HSCT. As other authors have published, activating receptors can be significantly upregulated in cytokine-stimulated NK cells. In our experience, overnight incubation with IL-15 overcomes this limitation, enhancing three times NK cytotoxicity, in vitro. Conclusion: The phenotype of NK cells and NK cytotoxicity ability are significantly altered early after allogeneic transplantation from unrelated donors using CD3/CD19-depleted graft. NK repertoire observed in patients was associated with the imbalance between CD56bright and CD56dim NK subsets and the expression of KIRs and NCRs. These data suggest a pattern consistent with an ongoing NK maturation after MUD and HSCT transplantation. In our experience, the phenotype and functional pattern of NK cells observed is suggestive of a cytokine-driven process. IL-15 stimulated NK cells could be helpful to optimize adoptive antitumor NK immunotherapy to enhance GvL effect as early as possible after transplantation. Disclosures: No relevant conflicts of interest to declare.

Mono/Oligoclonal T and NK Cells Are Common in Philadelphia Chromosome Positive (Ph+) Leukemia Patients at Diagnosis and Expand During Successful Tyrosine Kinase Inhibitor Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 856-856
Author(s):  
Anna Kreutzman ◽  
Vesa Juvonen ◽  
Veli Kairisto ◽  
Marja Ekblom ◽  
Leif Stenke ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Gene Rearrangements ◽  
Imatinib Treatment ◽  
Healthy Controls ◽  
Clonal Rearrangement

Abstract Abstract 856 Introduction. Central to current treatment of Ph+ leukemia patients are tyrosine kinase inhibitors (TKIs), which predominantly target the BCR-ABL1 kinase in malignant cells. However, broader-spectrum 2nd generation TKIs, such as bosutinib, dasatinib and nilotinib, also inhibit off-target kinases with important physiological functions. Several in vitro studies have implied that TKIs may have immunosuppressive effects by suppressing activation and proliferation of effector lymphocytes. In contrast, we recently observed immunostimulation during dasatinib therapy in the form of marked expansion of clonal cytotoxic lymphocytes (T- and NK cells) resulting in chronic LGL-type lymphocytosis in peripheral blood (PB). The prevalence, detailed molecular background and clinical implications of clonal lymphocytes during TKI therapy are currently unknown. The aim of this study was to comprehensively analyze clonality and evolution of lymphocyte clones during TKI therapy. Patients and methods. The study population included patients with Ph+ leukemia, both CML (n=28) and ALL patients (n=4) on dasatinib (n=23) and imatinib (n=9) therapies. In addition, samples from 12 healthy controls and diagnostic samples from the nine imatinib treated patients were analyzed. Lymphocyte clonality was determined by analysis of PB mononuclear cells (MNC) for clonal T cell receptor (TCR) γ and δ gene rearrangements by 18 primer pairs covering most known clonal TCR γ and δ rearrangements. Upon positive reaction in heteroduplex analysis, the purified PCR products were sequenced. If clonal rearrangement was observed, allele-spesific PCR primers were designed to allow for quantitative follow-up of lymphocyte clones in each patient. Results. Sequencing-confirmed clonal TCR γ rearrangement was observed only in 1 of 12 healthy controls and no TCR δ gene rearrangements were found in this group. Surprisingly, 7 of 9 (78%) CML patients showed clonal TCR rearrangements at diagnosis. In 3 patients the clonal rearrangement was detected in the TCR δ genes, in 7 patients in the TCR γ genes and 3 patients had rearrangemens both in TCR δ and γ genes. After one year of imatinib treatment the same clones could be detected in 5 of the 7 patients (71%). Although clonal cells were observed, none of the imatinib patients had signs of a concomitant lymphoproliferative disorder and the distribution of lymphocyte subclasses was normal. Next, 23 patients treated with dasatinib were studied, 10 without (LGLneg) and 13 with PB LGL lymphocytosis (LGLpos) including T- or NK-cell expansions. In all LGLpos dasatinib patients (including patients with a CD3neg NK-cell expansion) clonal TCR γ or δ rearrangements were found. In LGLneg dasatinib patients the prevalence of TCR rearrangements was 80%. LGLpos patients had more often clonal rearrangements in TCR δ genes (62%) than LGLneg patients (10%). No differences in clonal rearranged TCR γ genes (77% vs. 80%) were detected. Most patients displayed more than one clonal TCR rearrangement. Quantitative follow-up of LGLpos patients revealed that the expansion of a single predominant lymphocyte clone accounted for LGL lymphocytosis. Intriguingly, quantitative follow-up of lymphocyte clones by PCR showed that the observed clones existed at low levels already before start of dasatinib therapy during imatinib treatment, but no lymphocyte expansions were then seen. Sorting of lymphocytes showed that clonal cells resided in the CD8+ and CD4+ T-cell populations and, strikingly, also among CD16/56+CD3neg NK cells. All dasatinib patients with NK cell expansions (n=3) showed TCR δ rearrangements in their NK cells. Conclusions. Clonal lymphocytes could rarely be found in healthy controls. In contrast, they were frequently present in CML patients at diagnosis and persisted during TKI therapy. In a distinct subgroup of dasatinib treated patients, clonal cells massively expanded during successful therapy. Clonal TCR rearrangements were detected in CD4+, CD8+ and, unexpectedly, also in NK cells. The epitopes and function of clonal, CML-associated lymphocytes are under investigation. Previous studies showed that clonal expansions during dasatinib were associated with excellent, long-lasting therapy responses in advanced leukemia. We therefore hypothesize, that the clonal lymphocytes present at CML diagnosis may be anergic anti-leukemic cells and part of the immune escape mechanisms inherent to leukemogenesis and that dasatinib therapy can reverse this anergy. Disclosures: Ekblom: BMS: Honoraria. Seggewiss:BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:BMS: Honoraria.

Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1865-1865
Author(s):  
Inger S. Nijhof ◽  
Michel de Weers ◽  
Pascale Andre ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Monoclonal Antibody ◽  
Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

Molecular and Cytogenetic Response After 3 Months of Imatinib Treatment Is Predictive for the Risk of Disease Progression and Death in Newly Diagnosed Chronic Myeloid Leukemia Patients – a Follow-up Analysis of the German CML Study IV

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 783-783 ◽  
Author(s):  
Benjamin Hanfstein ◽  
Martin C. Müller ◽  
Philipp Erben ◽  
Michael Lauseker ◽  
Susanne Saussele ◽  
...  
Keyword(s):  
Overall Survival ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Risk Group ◽  
Cytogenetic Response ◽  
Imatinib Treatment ◽  
Risk Of Disease ◽  
Equity Ownership

Abstract Abstract 783FN2 Introduction: The advent of second generation tyrosine kinase inhibitors (TKI) in the front line treatment setting of chronic myeloid leukemia (CML) has tightened the evaluation of imatinib response. Early assessment of response markers might identify slow responders harboring a BCR-ABL positive clone with an inferior susceptibility to tyrosine kinase inhibition. This group of patients could benefit from an early dose escalation or a change of treatment to a second generation TKI thus avoiding the risk of disease progression. Therefore we sought to evaluate the impact of molecular and cytogenetic response levels after 3 months of imatinib treatment on the further course of disease. Patients and methods: A total of 1,340 patients (median age 52 years, range 16–88, 40% female) were included into the randomized German CML study IV and treated with an imatinib based therapy consisting of imatinib 400 mg/d (n=381), imatinib 800 mg/d (n=399) and combinations of standard dose imatinib with interferon alpha (n=402) and low-dose cytarabine (n=158). Median follow-up was 4.7 years (range 0–9). Molecular response after 3 months was assessed in 743 patients, cytogenetic response in 498 patients. The BCR-ABL expression was determined by quantitative RT-PCR and standardized according to the international scale (BCR-ABL IS). Only patients expressing typical BCR-ABL transcripts (b2a2, b3a2, b2a2 and b3a2) were considered. Cytogenetic response was determined by conventional metaphase analysis. Disease progression was defined by the incidence of accelerated phase, blastic phase or death from any reason. A landmark analysis was performed for progression free survival (PFS) and overall survival (OS). Results: Disease progression was observed in 149 patients (11.1%), 127 patients died (9.5%). After 3 months of treatment the median BCR-ABL IS was 2.6% (0-100), the median proportion of Philadelphia chromosome positive metaphases (Ph+) was 8% (0-100). The BCR-ABL landmarks of 1% and 10% after 3 months of imatinib both proved to discriminate significantly for PFS and OS: BCR-ABL IS <1% (n=233) vs. ≥1% (n=486), p=0.041 for PFS, p=0.048 for OS; BCR-ABL IS <10% (n=524) vs. ≥10% (n=195), p=0.004 for PFS and p=0.001 for OS. A stratification in 3 risk groups according to the achievement of a BCR-ABL IS of <1%, 1–10% and >10% after 3 months resulted in a significant difference between the poor risk group (>10%, n=195) and the intermediate risk group (1-10%, n=291): p=0.038 for PFS and p=0.012 for OS. The difference between the intermediate risk group and the good risk group (<1%, n=233) was not significant. The five year survival probability was 97%, 94% and 87% for the good, intermediate and poor risk group, respectively. Cytogenetic response landmarks after 3 months of imatinib were also predictive for PFS and OS: Ph+ ≤35% (n=362) vs. Ph+ >35% (n=123), p=0.022 for PFS, p=0.043 for OS; Ph+ ≤65% (n=401) vs. Ph+ >65% (n=84), p=0.004 for PFS and p=0.011 for OS. A 3 group stratification did not reach statistical significance. Conclusions: The achievement of molecular and cytogenetic response landmarks after 3 months of imatinib treatment is predictive for long term progression free and overall survival. At 3 months a BCR-ABL IS of 10% or more is associated with a 5-year overall survival of 87% suggesting an early change of treatment, whereas a BCR-ABL IS of 1% or less indicates a favorable 5-year overall survival of 97%. Disclosures: Schnittger: Münchner Leukämie Labor: Equity Ownership. Haferlach:Münchner Leukämie Labor: Equity Ownership. German CML Study Group:Deutsche Krebshilfe: Research Funding; Novartis: Research Funding; BMBF: Research Funding; EU: Research Funding; Roche: Research Funding; Essex: Research Funding.

Natural Killer (NK) Cell Reconstitution After Haploidentical Unmanipulated Bone Marrow Transplantation with Reduced Intensity Conditioning

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4557-4557 ◽  
Author(s):  
Isabel Gonzalez-Gascon y Marin ◽  
Ana Maria Perez-Corral ◽  
Jorge Gayoso ◽  
Javier Anguita ◽  
Cristina Pascual ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Median Number ◽  
Reduced Intensity Conditioning ◽  
Hematopoietic Stem ◽  
Reduced Intensity

Abstract Abstract 4557 BACKGROUND: Natural killer (NK) cells are innate immune effectors that directly lyse virally infected or malignant cells. There are 2 different subsets of NK cells with distinct phenotypic and functional characteristics: the CD56dim subset, which composes 90% of peripheral blood NK cells and has a cytotoxic function, and the CD56bright subset, which cooperates with dendritic cells and T cells in lymph nodes to secrete interferon and promote adaptive immune responses. NK cells are the first donor-derived lymphocyte subset to reconstitute after hematopoietic stem cell transplantation, reaching normal levels after 1 month. Nearly all phenotyping studies of NK subsets after haploidentical hematopoietic stem cell transplantation (HHSCT) reveal a rapid reconstitution of NK cells towards the CD56bright subset. In addition, Y.-J. Chang et al found the highest 2-year survival in patients with a high number of CD56bright NK cells after unmanipulated HHSCT. We analyzed reconstitution of the NK compartment between days 90 and 180 after unmanipulated bone marrow HHSCT with reduced intensity conditioning (RIC). METHODS: Six adults received unmanipulated bone marrow HHSCT after RIC (fludarabine 30 mg/m2 [day –6 to –2], cyclophosphamide 14.5 mg/kg [day –6 and –5], and busulfan i.v. 3.2mg/kg [day –3]) at our institution between July 2007 and July 2010. Prophylaxis for acute graft-versus-host disease (GvHD) consisted of cyclophosphamide 50mg/kg (days +3 and +4) and cyclosporine A and mycophenolate mofetil from day +5 onwards. We monitored the reconstitution kinetics of circulating NK cells (CD56+, CD3–), and the CD56bright and CD56dim subsets by multiparametric flow cytometry (FC 500 Beckman® Coulter) at day +90 and day +180 after transplantation. Patient characteristics and clinical outcomes are shown in Table 1. 6 patients who underwent allogeneic HLA-identical sibling HSCT with RIC during the same period were used as controls. RESULTS: After HHSCT, NK cells reached normal levels in all patients but one at day +90, with a median number of NK cells of 111/mm3 (range, 25–195/mm3). At day +180 the median number of NK cells was 92/mm3 (range, 4–272/mm3). When we analyzed the absolute number of CD56bright and CD56dim subsets at day +90, we observed 2 patterns: Two patients showed skewed NK cell reconstitution towards CD56bright (Patient no. 3: 54 CD56bright/mm3; 11 CD56dim/mm3. Patient no. 4: 70 CD56bright/mm3; 17 CD56dim/mm3). Three patients reconstituted with a CD56dim/CD56bright ratio towards the CD56dim cell subset, similar to that of healthy adults (Patient no. 1: 17 CD56bright/mm3; 178 CD56dim/mm3. Patient no. 5: 9 CD56brigh/mm3; 135 CD56dim/mm3. Patient no. 6: 20 CD56bright/mm3; 116 CD56dim/mm3). One patient did not achieve adequate NK cell reconstitution (Patient no. 2: 15 CD56bright/mm3; 10 CD56dim/mm3). In contrast, in the control group, an increase in the CD56bright NK cell subset was not observed in any of the patients at any point. It is worth noting that 2 of the 3 patients with better clinical outcome (no GvHD, no relapse), namely patients no. 3 and no. 4 were the ones with skewed NK cell reconstitution towards the CD56bright NK cell subset. The other patient with a better clinical outcome (patient no. 6) had a normal CD56dim/CD56bright ratio at day +90. However, he showed an early CD56bright reconstitution (363 CD56bright/mm3; 34 CD56dim/mm3) in an additional determination on day +30. NK cell subsets reconstitution kinetics is shown in Figure 1. CONCLUSIONS: In our experience, NK cell reconstitution is adequate after RIC unmanipulated bone marrow HHSCT. Some patients recovered with a high proportion of CD56bright NK cells, as previously reported in other studies on HHSCT. Although limited by the sample size, our results are consistent with the previously observed survival advantage of patients with high early levels of CD56bright NK cells after unmanipulated haploidentical transplantation. Disclosures: No relevant conflicts of interest to declare.

CSL362: A Monoclonal Antibody to Human Interleukin-3 Receptor (CD123), Optimized for NK Cell-Mediated Cytotoxicity of AML Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3598-3598 ◽  
Author(s):  
Samantha J. Busfield ◽  
Mark Biondo ◽  
Mae Wong ◽  
Hayley S. Ramshaw ◽  
Erwin M Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibody ◽  
Mouse Model ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Cell Leukemia ◽  
Interleukin 3

Abstract Abstract 3598 The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin's lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively. We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells. The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb. The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells. The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures: Busfield: CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

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