Human Flt3 Research Articles

Abstract 6313: Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia

Abstract Acute myeloid leukemia is a grievous illness. BiTE® (bispecific T cell engager) molecules redirect T cells by engaging CD3 and a tumor-associated antigen (TAA). These molecules have shown clinical efficacy but one mechanism of resistance is loss of a single TAA. We hypothesized that a BiTE® molecule targeting >1 TAA could reduce relapse frequency. FLT3 and CD123, were selected for a dual-targeting BiTE® (dBiTE࣪) molecule.A half-life extended (HLE) CD123-FLT3 dBiTE࣪ molecule was evaluated in vitro, in mouse xenografts, and in non-human primate (NHP) tolerability studies. The molecule had nM affinity for human and NHP FLT3, CD123, and CD3, and pM efficacy in cytotoxicity assays using human T cells or NHP PBMCs. The molecule achieved 100% killing against single-positive (sp) cells (CRISPR-generated isogenic cell lines) at potencies like those of double-positive (dp) cells (7.4 ±. 4.1 pM FLT3sp, 7.1 ±. 3.8 pM CD123sp and 3.4 ±. 1.5 pM dp, n=3). In a mouse xenograft model, the molecule induced significant activity and extended survival > 3 weeks (1.0, 0.1, and 0.01 mg/kg (p ≤ 0.001)). In mice with sp tumors, survival benefit could not be calculated due to high survival, demonstrating that both arms of the molecule are active. In NHP, the molecule had a half-life of 52 hours (0.3 or 3 µg/kg). FLT3 mRNA levels, a marker of FLT3-expressing cells, decreased in blood following dosing. Repeat dosing was not tolerated, and cytokine release was observed. Some cytokines were reduced while others increased. CD123 is reportedly expressed on endothelial cells (ECs), with increased expression in inflammatory conditions. An immunohistochemical survey found that CD123 is expressed on human and NHP monocyte/macrophages and ECs with limited distribution in lymphoid tissues and lamina propria of the gut. We hypothesized that cytokine release following administration may result in increased CD123 expression and in turn, further increased cytokine levels with repeat dosing. CD123 was detected on primary human umbilical vein ECs (HUVECs). In co-cultures of HUVECs and T cells, a CD123 BiTE® molecule induced expression of CD123 on HUVEC cells at concentrations that elicited redirected lysis (12.5 pM), T cell activation, and cytokine secretion. To better understand BiTE®-induced upregulation of CD123 on ECs, HUVECs were cultured with supernatants (SN) from a TDCC assay or recombinant IL-3, IL-6, TNFα, or IFNγ. Assay SN and TNFα induced >2-fold CD123 expression on HUVECs, but not on CD123-negative primary human pulmonary microvascular ECs. These data demonstrate that CD123 expression on ECs was increased upon exposure to a CD123 mono-targeting BiTE® molecule, potentially through BiTE®-induced secretion of TNFα. Additional studies are ongoing.In sum, a CD123-FLT3 HLE dBiTE࣪ molecule was active against both dp- and sp-positive target cells in vitro and in vivo. Careful selection of TAA for dBiTE࣪ molecules is necessary to increase efficacy and maintain safety. Citation Format: Rebecca Goldstein, Christine Karbowski, Anja Henn, Petra Deegen, Joachim Wahl, Katja Matthes, Christoph Dahlhoff, Brooke Rock, Sabrina Benchaar, Katie Hsu, Brandy Alexander, Matthias Friedrich, Joan Lane, Xiaoting Wang, Jonas Anlahr, Markus Muenz, Tobias Raum, Tara L. Arvedson. Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6313.

Abstract 913: FLT3-targeting TriTACs are T cell engagers for treatment of acute myeloid leukemia

Abstract FLT3 (fms related receptor tyrosine kinase 3) is a cell surface protein expressed in hematopoietic stem and progenitor cell populations1. FLT3 RNA is overexpressed in greater than 95% of acute myeloid leukemia (AML) samples2, and FLT3 mutations are found in 25% to 35% of AML samples1. In 2020 in the United States, an estimated 19,940 new AML cases and 11,180 AML-related deaths are expected3, exemplifying that better therapies are needed to treat this disease. Given the frequency of FLT3 expression in AML and unmet medical need for patients, FLT3 represents an attractive target for T cell-redirecting immunotherapy in AML. FLT3-targeting Tri-specific T cell Activating Constructs (FLT3 TriTACs) are designed to simultaneously engage FLT3 on a target AML cell and CD3 on a T cell, resulting in T cell activation, proliferation, and lysis of the AML cell. FLT3 TriTACs are ~53 kDa molecules that consist of three binding domains: an N-terminal single domain antibody (sdAb) that binds to human FLT3, a middle sdAb that binds to human serum albumin (HSA) to extend the half-life, and a C-terminal single chain Fv (scFv) that binds to CD3ϵ of the T cell receptor (TCR). Biophysical analyses of these molecules indicate they are stable with high monomer content. FLT3 TriTACs bind to human and cynomolgus monkey FLT3, CD3ϵ and albumin with similar affinities. Flow cytometry analysis of T cells from various normal donors and a panel of FLT3-positive and FLT3-negative tumor cell lines confirmed binding of FLT3 TriTACs to their targets expressed on the cell surface. FLT3 TriTACs induce potent killing of FLT3-expressing AML cell lines in vitro. In co-cultures of T cells from normal human donors, target tumor cells, and HSA, these TriTACs mediated dose-dependent and FLT3-dependent cytotoxicity. Cell lines engineered to express cynomolgus FLT3 were also readily killed by T cells with FLT3 TriTACs. Preclinical evaluation of FLT3 TriTACs in vitro, in mouse tumor models, and cynomolgus monkey pharmacokinetic studies will be presented.

Evaluation of a FLT3 Bite® for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a disease with high unmet medical need. Standard induction chemotherapy can lead to complete remission in patients with a favorable genetic risk profile; however, about half of patients are ineligible for this intensive regimen. In those patients with a non-favorable genetic risk profile, the preferred treatment strategy is allogeneic stem cell transfer, but the majority of patients are ineligible due to age and other comorbidities.FLT3 is a receptor tyrosine kinase that induces cell growth in myeloid progenitor cells. Activating mutations in FLT3 have been identified in approximately 30% of AML patients. Expression of mutant FLT3 and overexpression of wild type FLT3 are associated with poor prognosis in AML, leading to clinical investigation of targeted FLT3 inhibitors, including midostaurin which was recently approved for patients with newly diagnosed FLT3-mutant AML. These inhibitors are likely to be effective only in certain patient subpopulations and may ultimately give rise to acquired resistance. To drive cures in AML, effective therapies for broad patient populations that can eliminate residual disease are needed.Bispecific T cell engaging antibody constructs (BiTE®) are a class of immunotherapy agents which redirect T lymphocytes to a specific antigen expressed on tumor cells. Blinatumomab, a BiTE® containing a single chain variable fragment (scFv) that binds CD19 on B lymphocytes and an scFv that binds CD3 on T lymphocytes, has shown clinical efficacy for treating CD19-positive hematological malignancies such as ALL. To extend the serum half-life, next generation BiTE® antibody constructs have been modified to contain Fc-moieties, allowing for weekly dosing.To evaluate FLT3 as a BiTE® target in AML, we assessed expression of FLT3 on primary AML patient blasts using flow cytometry. Over 83% of AML patients (n = 192) were FLT3-positive (MFI ratio > 1.5) with similar expression in bulk and leukemic stem cells. A half-life extended FLT3 BiTE® (FLT3 HLE BITE®) was generated and evaluated in vitro as well as in vivo in a mouse xenograft tumor model and non-human primates (NHP). FLT3 HLE BiTE® had subnanomolar binding affinity for human FLT3 and nanomolar binding affinity for human CD3 and NHP FLT3 and CD3. In cytotoxic assays targeting human AML cell lines, FLT3 HLE BiTE® exhibited picomolar efficacy using as effector cells human pan T cells in a 10:1 E:T ratio (range: 2 to 10 pM) or NHP PBMCs in a 5:1 E:T ratio (range: 30 to 80 pM). In mice injected with FLT3-positive MOLM13 cells and human CD3-positive T cells, FLT3 HLE BiTE® induced robust anti-tumor activity and extended survival ≥ 2-fold at all doses (n = 10 per group, p ≤ 0.0015). Following a single 5 µg/kg dose in NHP (n=2), FLT3 HLE BiTE® exhibited a reproducible pharmacokinetic profile with a 66 hour half-life. In this study, we observed hallmarks of BiTE® activation such as induction of IL-6 and MCP-1 and indication of a pharmacodynamic effect through increased soluble FLT3 ligand. Following repeat-dosing with FLT3 HLE BiTE®, digital droplet PCR was used to measure FLT3 transcript in NHP blood and bone marrow. Robust depletion of FLT3+ cells was observed in both NHP blood (≥ 95% reduction) and bone marrow (≥ 68% reduction) at all doses (5 µg/kg, 25 µg/kg, 75 µg/kg, n = 3/group). Taken together, FLT3 HLE BiTE® has potent in vitro and in vivo activity and may provide therapeutic benefit to AML patients. DisclosuresGoldstein:Amgen: Employment. Henn:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Archibeque:Amgen: Employment. Frank:Amgen Inc.: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Raum:Amgen Research (Munich): Employment. Li:Amgen, Inc.: Employment. Wahl:Amgen Research (Munich): Employment. Rock:Amgen, Inc.: Employment, Equity Ownership. Thomas:Amgen Research (Munich): Employment. Karbowski:Amgen, Inc.: Employment. Krupka:Amgen Research (Munich): Research Funding; Amgen, Inc: Research Funding. Subklewe:Amgen Research (Munich): Research Funding; Amgen, Inc.: Research Funding. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

A Phase I/II Study of Selinexor (SEL) with Sorafenib in Patients (pts) with Relapsed and/or Refractory (R/R) FLT3 Mutated Acute Myeloid Leukemia (AML)

Background: Responses to FLT3-inhibitors are usually transient due to emergence of resistance through the acquisition of kinase domain point mutations and other non-mutational mechanisms. SEL is a potent first-in-class Selective Inhibitor of Nuclear Export/SINE™ that exerted marked cell killing of human and murine FLT3 -mutant AML cells, including those with ITD, D835Y, ITD+Y842C or ITD+F691L mutations by modulating the cdk inhibitor p27 and anti-apoptotic Mcl-1 (Zhang W et al, Blood 2015 126). The combination of SEL+sorafenib had synergistic pro-apoptotic effects in FLT3-mutated AML cells by suppressing phosphorylation levels of FLT3 and its downstream signaling mediators ERK/AKT, and by inducing myeloid differentiation in ITD and D835 mutated cell lines.Methods: We designed a phase I/II trial of SEL with sorafenib for FLT3-ITD and/or -D835 R/R AML pts. In phase I, the primary objectives were to determine the maximum tolerated dose (MTD), the recommended phase 2 dose (RP2D), and dose-limiting toxicity (DLT) of the combination. In phase II, primary objectives included the rate of complete remission (CR) + CR with incomplete count recovery (CRi) + partial response + >50% blast reduction within 3 months of therapy initiation. Secondary objectives were safety and overall survival (OS). SEL was given orally twice a week for 3-weeks on and 1-week off in 28-day cycles and sorafenib was given continuously at a dose of 400mg twice daily from cycle 1 day 1 of SEL. In phase I, SEL dose was de-escalated in “3+3” fashion starting at dose level 0 and going down if DLTs were exceeded (dose levels 0, -1, -2 = 80, 60, 40 mg, respectively). Once MTD was established, the phase II enrolled pts in 2 cohorts: prior FLT3-inhibitor failure (cohort 1) and FLT3-inhibitor naïve (cohort 2). PB or BM samples were collected at pre-dose (C1D1), 24 h (C1D2) and day 28 (C1D28) of cycle 1, and apoptosis induction was determined by measuring annexin V positivity with FACS.Results: Fourteen pts with a median age of 71 years (range, 24-81) were enrolled. All pts had baseline next generation sequencing for AML specific mutations (Figure 1A). The median number of prior therapies was 3 (range, 1-5) as follows: salvage (S) 1: n=1, S2: n=5, S3+: n=8. 11 (79%) pts had prior FLT3-inhibitors: 1 prior FLT3-inhibitor (n=9); 2 prior FLT3-inhibitors: n= (2). Three pts had prior SCT. Four pts were treated at dose level 0 (SEL 80mg) with DLTs in 2 (Grade (G) 3 sepsis, n=1; G3 mucositis, syncope, adrenal insufficiency, n=1). Three pts were subsequently treated at dose level -1 (SEL 60mg) with no DLTs and this was established as the MTD/RP2D. Seven pts were treated in expansion: 6 had prior FLT3-inhibitor therapy (cohort 1) and 1 had no prior FLT3-inhibitor (cohort 2).The median duration of treatment for all 14 pts was 28 days (range, 14-122) and therapy is ongoing in 1 pt. All pts are evaluable for response. CRp/CRi and >50% blast reduction as best responses were seen in 4/14 (29%) and 2/14 (14%) pts, respectively. All 6 responders had a prior FLT3 inhibitor yielding a response rate of 55% (6/11) pts with prior FLT3 inhibitor therapy. 3 pts with CRp/CRi (2 with FLT3-ITD and 1 with ITD+D835) achieved negative RT-PCR for FLT3. (Figure 1B). One pt in CRi was bridged to SCT and remains alive at 1 year. The most common G 3/4 adverse events irrespective of causality were bleeding (gastrointestinal, n=4; intracranial, n=1), febrile neutropenia (n=4), pneumonia (n=3) and syncope (n=2). The reasons for discontinuing study therapy were disease progression (n=9; 64%), toxicity (n=3; 21%) and SCT (n=1; 7%). The median OS for pts on study was 3.5 months (range, 0.9-15). Translational analysis showed that SEL+sorafenib induced apoptosis in 4 of 5 tested patients, especially in the BM samples (Figure 1C)Conclusions: The combination of SEL and sorafenib is safe with clinical activity and apoptosis induction in R/R FLT3+ AML. The benefit was especially encouraging in pts exposed to prior FLT3 inhibitors, with a response rate of 55% (6/11; including 2 CRi and 2 CRp). The RP2D is 60 mg twice weekly of SEL and the study is accruing (NCT01607892). [Display omitted] DisclosuresDaver:Kiromic: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Incyte Corporation: Honoraria, Research Funding; Pfizer Inc.: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. DiNardo:Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Cortes:BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; ARIAD: Consultancy, Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding. Kantarjian:Novartis: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding.

Functional and Phenotypic Characterization of Cord Blood and Bone Marrow Subsets Expressing FLT3 (CD135) Receptor Tyrosine Kinase

AbstractThe class III receptor tyrosine kinase FLT3/FLK2 (FLT3; CD135) represents an important molecule involved in early steps of hematopoiesis. Here we compare cell-surface expression of FLT3 on bone marrow (BM) and cord blood (CB) cells using monoclonal antibodies (MoAbs) specific for the extracellular domain of human FLT3. Flow cytometric analysis of MACS-purified BM and CB cells showed that 63% to 82% of BM CD34+ and 88% to 95% of the CB CD34+ cells coexpress FLT3. Clonogenic assays and morphological characterization of FACS-sorted BM CD34+ cells demonstrate that colony-forming unit–granulocyte-macrophage (CFU-GM) and immature myelo-monocytic precursor cells are enriched in the subpopulation staining most brightly with the FLT3 MoAb whereas the majority of the burst-forming units-erythroid (BTU-E) and small cells with lymphoid morphology are found in the FLT3− population. In contrast, statistically indistinguishable proportions of CFU-granulocyte-erythrocyte-megakaryocyte-macrophage (CFU-GEMM) and more primitive cobblestone area forming cells (CAFC) were detected in both fractions, albeit the FLT3+ fraction consistently showed more CAFC activity than the FLT3− fraction. Although in both, BM and CB the majority of CD34+CD117+ (KIT+), CD34+CD90+ (Thy-1+), and CD34+CD109+ cells coexpress FLT3, three-color phenotypic analyses are consistent with the functional findings and suggest that the most primitive cells defined as CD34+CD38−, CD34+CD71low, CD34+HLA-DR−, CD34+CD117low, CD34+CD90+, and CD34+CD109+ express low levels of cell-surface FLT3 and were therefore not enriched to a statistically significant extent with the bright versus negative sorting scheme. Thus, clear segregation of the most primitive progenitors from BM CD34+ cells was confounded by low apparent levels of FLT3 cell-surface expression on these cells, whereas myeloid progenitors unambiguously segregated with the FLT3 brightest cells and erythroid progenitors with the FLT3 dimmest. Additional phenotypic analyses using MoAbs against progenitor/stem cell markers including the mucinlike molecule MGC-24v (CD164), the receptor tyrosine kinases TIE, FMS (CD115), and KIT (CD117) further illustrate the differences in surface antigen expression profiles of BM and CB CD34+cells. Notably, CD115 is rarely detected on CB CD34+cells, whereas 20% to 25% of the BM CD34+FLT3+ cells are CD115+. Furthermore, 80% to 95% of the CB CD34+CD117+ but only 60% to 75% of the BM CD34+CD117+ cells coexpress FLT3. Only a negligible amount of CD34+CD19+ are detected in CB, while in BM 20% to 30% of CD34+CD19+ presumed pro/pre-B cells coexpress FLT3. In contrast, the majority of the CD34+CD164+ and CD34+TIE+ subsets in both CB and BM coexpress FLT3. Analysis of unseparated cells showed that FLT3 expression is not restricted to CD34+ subsets. About 65% to 70% of lymphocyte-gated BM CD34−FLT3+ cells are positive for the monocytic marker CD115 whereas 25% to 30% of these cells consist of CD10 expressing B-cell precursors. Finally, CD34−monocytes in BM, CB, and PB express FLT3 whereas granulocytes are FLT3−. Our data show that detectable FLT3 appears first at low levels on the surface of primitive multilineage progenitor cells and disappears during defined stages of B-cell development, but is upregulated and maintained during monocytic maturation. © 1997 by The American Society of Hematology.

First-in-Human Phase I Dose Escalation and Expansion Study Evaluating the Fc Optimized FLT3 Antibody Flysyn in Acute Myeloid Leukemia Patients with Minimal Residual Disease

Even when achieving morphological complete remission (CR) after induction therapy, roughly half of acute myeloid leukemia (AML) patients display measurable residual disease (MRD) and eventually relapse. The surface receptor FLT3/CD135 is expressed on AML cells in almost all patients and constitutes a highly selective target antigen for immunotherapy, as expression on healthy tissues is limited to low levels on dendritic cells, monocytes and hematopoietic progenitor cells. FLYSYN is a chimeric Fc-optimized IgG1 antibody that binds specifically and with high avidity to human FLT3 (CD135). Here we report updated results of an open-label, single-arm, first in man multicenter trial (recruitment March 2017 to March 2020) evaluating safety/tolerability and preliminary efficacy of FLYSYN in patients with AML (NCT02789254). Morphological CR with stable or increasing MRD in two sequential measurements using central RT-qPCR and/or next generation sequencing (NGS) constituted the main inclusion criterion. FLYSYN was administered i.v. over 3 h as single application in cohorts 1-5 (0.5 mg/m², 1.5 mg/m², 5 mg/m², 15 mg/m², 45 mg/m²); in cohort 6, 15 mg/m² were applied on day 1, 15 and 29. Three patients were treated per cohort except for cohorts 4 and 6, which were expanded to comprise 9 and 10 patients, respectively. Molecular response was defined as >1 log MRD reduction or negativity in bone marrow (BM). In total, 31 patients (median age 58 years; range, 21-80 years; male:female ratio: 1:1.8) were enrolled, of which 27, 3 and 1 were MRD-positive for mutated NPM1, mutated IDH2 and RUNX1-RUNX1T1, respectively. Based on pharmacokinetic analysis, the half-life of FLYSYN was estimated to be 6.5 days. In 8 patients (26%), a transient decrease of neutrophil count (2 adverse events (AEs) grade 3, others ≤ grade 2) was observed. No relevant effect on stem cell reserve as assessed by colony forming unit assays was detected in the so far analyzed 26 patients. No other AE > grade 2 or dose-limiting toxicity were observed. The most frequent AEs were unspecific and comprised fatigue and flu like symptoms (12%), musculoskeletal symptoms (8%) and laboratory abnormalities (42%). With regard to efficacy, molecular response to treatment was achieved in 11/31 patients (35%), with so far two patients achieving MRD negativity documented one year after treatment. Among the patients receiving 45 mg/m2 FLYSYN (in total, upon single or repetitive dosing), objective responses were achieved in 46% (6/13) cases, whereas 28% (5/18) responded to treatment with lower doses. Together, the results of our phase I trial demonstrate that FLYSYN is safe and very well tolerated as monotherapy in AML patients with molecular MRD. Early efficacy data are promising and warrant further evaluation in an up-coming phase II clinical trial. Disclosures Heuser: PriME Oncology: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Stemline Therapeutics: Consultancy; Janssen: Consultancy; Astellas: Research Funding; Roche: Research Funding; BerGenBio ASA: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Amgen: Research Funding; Karyopharm: Research Funding; Abbvie: Consultancy. Thol:Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kapp-Schwoerer:Jazz Pharmaceuticals: Honoraria, Research Funding. Grosse-Hovest:Synimmune: Current Employment. Steiner:Synimmune: Current Employment. Schlenk:Roche: Research Funding; Novartis: Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; PharmaMar: Research Funding; AstraZeneca: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Salih:Pfizer: Consultancy; Medigene: Consultancy; Novartis: Consultancy; Philogen: Consultancy; Synimmune: Consultancy, Research Funding.

Theoretical Studies Aimed at Finding FLT3 Inhibitors and a Promising Compound and Molecular Pattern with Dual Aurora B/FLT3 Activity.

FLT3 and dual Aurora B/FLT3 inhibitors have shown relevance in the search for promising new anticancer compounds, mainly for acute myeloid leukemia (AML). This study was designed to investigate the interactions between human FLT3 in the kinase domain with several indolin−2-one derivatives, structurally similar to Sunitinib. Molegro Virtual Docker (MVD) software was utilized in docking analyses. The predicted model of the training group, considering nineteen amino acid residues, performed in Chemoface, achieved an R2 of 0.82, suggesting that the binding conformations of the ligands with FLT3 are reasonable, and the data can be used to predict the interaction energy of other FLT3 inhibitors with similar molecular patterns. The MolDock Score for energy for compound 1 showed more stable interaction energy (–233.25 kcal mol−1) than the other inhibitors studied, while Sunitinib presented as one of the least stable (–160.94 kcal mol−1). Compounds IAF70, IAF72, IAF75, IAF80, IAF84, and IAF88 can be highlighted as promising derivatives for synthesis and biological evaluation against FLT3. Furthermore, IAF79 can be considered to be a promising dual Aurora B/FLT3 inhibitor, and its molecular pattern can be exploited synthetically to search for new indolin−2-one derivatives that may become drugs used in the treatment of cancers, including AML.

Abstract C083: Profiling of PDK1 inhibitor SNS-510 shows potent activity in hematologic and solid tumor cancer models and identifies CDKN2A-mutated cancers as potential targets with enhanced sensitivity

Abstract Background: Phosphatidylinositol (PI) dependent kinase 1, PDK1, is a master kinase that activates kinases important in cell growth and survival including members of the AKT, PKC, RSK and SGK families. PDK1 can interact with its substrates through PI-dependent or PI-independent (PIF-mediated) mechanisms. SNS-510 is a potent inhibitor of active and inactive conformations of PDK1 and binds deep in the adaptive pocket. This interaction perturbs the PIF-pocket, thereby affecting both PI-dependent and -independent substrates (Hansen Mol Cancer Ther 2015). SNS-510 is also a potent FLT3 inhibitor. Methods and Results: SNS-510 activity was evaluated in 320 OncoPanel™ cell lines derived from 20 different cancer types and exposed for 72 hours to a range of drug concentrations (10 µM-1 nM). SNS-510 showed strong and consistent anti-proliferative activity (overall median IC50 537 nM; range 14 nM-10 µM). Cell lines with IC50 <250 nM were classified as sensitive to SNS-510, while those with IC50 >2 μM were classified as resistant. Among the 59 cell lines classified as sensitive, the most prevalent tumor origin was lymphoma followed by leukemia, kidney and lung cancer. The AML FLT3-ITD positive cell line MV4-11 was the most sensitive cell line across the entire OncoPanel™ (IC50 14 nM). Univariate analysis comparing drug response data and public genomics data for the OncoPanel™ cell lines revealed potential biomarkers associated with sensitivity and resistance to SNS-510. The presence of mutations or deletions in the CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) gene was the genomic feature most significantly associated with sensitivity to SNS-510 (p=4.2E-05). Deletions in C9orf53 (CDKN2A divergent transcript), CDKN2B (Cyclin Dependent Kinase Inhibitor 2B) and CDKN2BAS (CDKN2B Antisense RNA 1) genes were also associated with significant sensitivity to SNS-510. Twenty-six (44%) sensitive cell lines were carriers of a CDKN2A mutation or deletion while only 7 (12%) resistant cell lines had a genetic defect in CDKN2A. We previously demonstrated (Hansen Mol Cancer Ther 2015) PDK1/RSK2/P70S6K/AKT pathway modulation and sustained antitumor effects in a human AML MV4-11 FLT3-ITD xenograft mouse model after SNS-510 administration PO. We have now evaluated SNS-510 antitumor activity in a human AML MOLM-16 FLT3 wild-type xenograft mouse model. Potent and dose-dependent tumor growth inhibition (range 52-97%) was observed in the MOLM16 model at SNS-510 PO dose levels of 10, 25 and 50 mg/kg qdx21. The antitumor efficacy was correlated with a profound drug-induced decrease in phospho-PDK1 (Ser241), phospho-AKT (Thr308) and phospho-RSK2 (Ser227) in MOLM16 tumor lysates. Conclusion: SNS-510 is a potent, orally bioavailable PDK1 inhibitor with broad activity in both hematologic and solid tumor cancers. SNS-510 inhibition of PDK1-signaling correlates with antitumor activity at tolerated doses and schedules in both FLT3-ITD and FLT3 wild-type AML xenograft mouse models. Profiling (anti-proliferative and molecular) in the OncoPanel cancer cell line screen identified CDKN2A mutated tumors as particularly sensitive to SNS-510, supporting a potential role by PDK1 inhibitors in combination with CDK4/6 inhibitors to reverse resistance and improve activity (Jansen Cancer Res 2017). Citation Format: Pietro Taverna, Stig Hansen, Jeff Iwig, Gene Jamieson, Judy A Fox. Profiling of PDK1 inhibitor SNS-510 shows potent activity in hematologic and solid tumor cancer models and identifies CDKN2A-mutated cancers as potential targets with enhanced sensitivity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C083. doi:10.1158/1535-7163.TARG-19-C083

Interim Results of a First in Man Study with the Fc-Optimized FLT3 Antibody Flysyn for Treatment of Acute Myeloid Leukemia with Minimal Residual Disease

Background: Substantial surface expression of the FLT3 receptor can be measured on blast cells in 70% to 100% of acute myeloid leukemia (AML) patients, while no or only low levels are expressed on healthy cells like monocytes and progenitor stem cells. Thus, FLT3 is a suitable and highly selective target for therapeutic antibodies. FLYSYN is a chimeric Fc-optimized IgG1 antibody and binds specifically and with high avidity to human FLT3 (CD135). Despite achievement of complete remission, roughly half of AML patients display minimal residual disease (MRD) after end of therapy and relapse. Methods: We perform an open-label, single-arm, first in man multicenter trial to assess safety and tolerability as well as preliminary efficacy of FLYSYN as monotherapy in adult (≥18 years) AML patients in complete remission with MRD (NCT02789254). FLYSYN is administered as IV infusion over a 3 hour period. Recruitment started in March 2017 with an estimated maximum number of 31 patients and estimated recruitment until January 2020. The main inclusion criterion was confirmed stable or increasing MRD positivity in two sequential measurements. MRD was measured by central RT-qPCR and/or next generation sequencing (NGS). Sensitivity with RT-qPCR (for NPM1 only) was 10-6 and 10-4 with NGS. Patients with acute promyelocytic leukemia as well as prior hematopoietic stem cell transplantation were excluded. Using a "3 + 3" dose escalation design, five of the planned six cohorts with escalating doses have currently completed treatment (cohort 1: 0.5 mg/m² BSA; cohort 2: 1.5 mg/m² BSA; cohort 3: 5 mg/m² BSA; cohort 4: 15 mg/m² BSA; cohort 5: 45 mg/m²; cohort 6: in total 45 mg/m²; 15 mg/m² on day 1, 15 and 29). Three patients were treated per cohort, except for cohort 4, which was expanded to nine patients. The interim analysis for preliminary efficacy was performed after 18 patients were treated in cohorts 1-4. Response is defined as 1 log MRD reduction. Results: Median age was 60 years (range, 21-80 years). Sixteen patients were MRD positive for NPM1 and one patient each for RUNX1-RUNX1T1 and IDH2. So far, FLYSYN was well tolerated. Only one temporary grade 3 adverse event (AE) occurred (neutrophil decrease on day 3 only) in a patient of cohort 3, which was suspected to be related to FLYSYN treatment. There were no reported dose-limiting toxicities. The most frequently reported AEs were grade 1 and 2 gastrointestinal toxicities and laboratory abnormalities, which all were manageable with supportive care. After treatment, neither human anti-mouse nor anti-human antibodies were detected in any of the patients. Preliminary pharmacokinetic analysis was performed in the first 12 patients of the study and revealed a half-life of FLYSYN of roughly 6.5 days. Regarding preliminary efficacy, 1 patient of cohort 1 achieved permanent MRD negativity in bone marrow (BM; lasting until day 545) and 1 patient a temporary BM MRD reduction (at day 15). One patient of cohort 2 achieved BM MRD negativity (day 22) with MRD progression on day 365, whereas the other 2 patients were BM MRD progressive. None of the patients of cohort 3 achieved a MRD response in BM, but 2 patients achieved a temporary MRD response in peripheral blood. Four patients of cohort 4 achieved non-permanent BM MRD negativity. Overall, 6 patients achieved BM MRD negativity (33 %, 6/18), enduring more than 1 year in 1 patient. Conclusions: Our data suggest that FLYSYN is safe and very well tolerated as monotherapy in MRD positive AML patients. Preliminary efficacy data are promising, and recruiting is ongoing in cohort 6 in which 15 mg/m² FLYSYN is given for three times. Up-dated results will be presented at the ASH meeting. Disclosures Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Steiner:Synimmune: Employment, Other: shareholder interest. Grosse-Hovest:SYNIMMUNE: Employment, Other: shareholder interest. Jung:Synimmune: Other: shareholder interest. Salih:SYNIMMUNE: Consultancy, Research Funding.

Semisynthetic Derivatives of Fradcarbazole A and Their Cytotoxicity against Acute Myeloid Leukemia Cell Lines.

Fourteen derivatives of the marine-derived fradcarbazole A were synthesized from staurosporine. Their structures were identified by NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS). The derivatives were screened in vitro for antiproliferative activity against three human leukemic cell lines (MV4-11, HL-60, K562). All of the derivatives displayed cytotoxicity against the human FLT-3 internal tandem duplication (ITD) mutant acute myeloid leukemia (AML) cell line MV4-11 with IC50 values of 0.32-0.96 μM. The mechanism of action studies indicated that the most effective 3-chloro-5‴-fluorofradcarbazole A (6) induced apoptosis of the MV4-11 cells and arrested the cell cycle at the G0/G1 phase. Furthermore, compound 6 can reduce the expression of FLT-3, CDK2, and c-kit. The results suggest that 3-chloro-5‴-fluorofradcarbazole A (6) is a potential candidate for developing novel anti-AML agents in the future.

Abstract 3217: Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA

Abstract Recent studies have highlighted the critical role of CD103+/CD141+ dendritic cells (DCs) in antitumor immunity. CDX-301 is a soluble, recombinant human FLT3 ligand (Flt3L). Flt3L is a hematopoietic cytokine which stimulates the proliferation and differentiation of various blood cell progenitors, including CD103+/CD141+ DCs. CDX-301 is in clinical development for multiple cancers and may hold significant opportunity for synergistic development in combination with other immunotherapies, in particular toll-like receptor 9 (TLR9) agonists that are known to promote maturation and activation of DCs. AST-008 is a TLR9 agonist oligonucleotide in a spherical nucleic acid (SNA) format and is in clinical development for multiple cancers in combination with pembrolizumab. SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. AST-008 induces potent TH1-type immune responses in vitro, in mice and non-human primates, and has shown potent antitumor activity as a monotherapy and enhanced checkpoint inhibitor activity in several murine tumor models. AST-008 increases tumor-infiltrating lymphocytes, interferon-inducible gene expression, and activation and expansion of CD8+ T cells with reduced T-regulatory cells in the tumor microenvironment. We examined the effects of the combination of CDX-301 and a murine version of AST-008, muAST-008, in mice on DCs and on antitumor efficacy in a tumor model. Mice were implanted with MC38 murine colon adenocarcinoma tumor cells (day 0) and animals were treated with either CDX-301 (5 μg, days 2-8 i.p.) only, muAST-008 only (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30 p.t.), or the combination of CDX-301 (5 μg, days 2-8) with muAST-008 (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30). Treatment with CDX-301 and muAST-008 showed an additive effect in retarding tumor growth and prolonging survival. To help understand the mechanisms involved in the antitumor effects, animals were treated with a similar course of CDX-301 and muAST-008 and spleens were harvested for flow cytometry evaluation 2 days after the last CDX-301 dose. For these studies, we also included a second DC activating agent, an agonist anti-CD40 mAb FGK45.5 (50 μg i.p. together with the last dose of CDX-301) among the combinations. We observed a significant increase in the percentage of CD103+CD8+ cDCs by the addition of muAST-008 to CDX-301 treatment. In addition, muAST-008 led to the up-regulation of activation markers on dendritic cells, which was markedly enhanced when combined with CD40 activation. These data demonstrate that muAST-008 leads to systemic activation of CDX-301 expanded DCs, leading to more potent anti-tumor immunity and support the potential of combining CDX-301 and AST-008 in augmenting the immunotherapy of cancers. Citation Format: Lawrence J. Thomas, Li-Zhen He, Lauren E. Gergel, Eric M. Forsberg, Elizabeth Q. Do, James M. Boyer, April R. Baronas, Mallary Rocheleau, Kathleen M. Borrelli, Anna Wasiuk, Jeffrey Weidlick, Henry C. Marsh, Bart R. Anderson, SubbaRao Nallagatla, Richard Kang, Ekambar R. Kandimalla, Tibor Keler. Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3217.

A Novel Full Length Anti-FLT3 CD3 Bispecific Antibody for the Treatment of Acute Myeloid Leukemia

FLT3 is a receptor tyrosine kinase expressed on the surface of acute myeloid leukemia (AML) patient blasts. FLT3 is the most frequently mutated gene in AML patients, and these mutations are associated with poor prognosis. Despite the development of small molecule inhibitors of FLT3 function and neutralizing FLT3 antibodies, there remains a need for antibodies that target the broad AML patient population with improved efficacy and safety.We chose to use Pfizer's proprietary full length humanized CD3 bispecific IgG molecule platform. The first step in the development was finding a suitable targeting epitope on FLT3 as not all epitopes result in optimal T cell activation in the context of CD3 binding. Through a combination of in vitro and in vivo studies, FLT3 antibodies targeting extracellular domain 4 of FLT3 were found to be more effective at AML cell depletion than other domains in the full-length bispecific IgG format, significantly outperforming the antibodies targeting the most membrane proximal region of domain 5. The final candidate antibody was engineered to have picomolar affinity for recombinant human FLT3 (<50 pM) to ensure that low levels of FLT3 present on AML (<5,000 per cell) could efficiently trigger T cell cytotoxicity.Anti-tumor activity of the final molecule was further characterized in vitro and in vivo. Three AML cell lines, Eol-1, Molm-13 and MV-411 express high, medium and low levels of FLT3, respectively, and were targeted by activated healthy donor T cells in the presence of the FLT3 bispecific with an efficiency that correlated with FLT3 surface density (EC50 range was 0.5 pM to 40 pM at E:T ratios of 1:1). Ability to target primary AML cells was confirmed in assays with primary AML blasts and autologous patient T cells (EC50s 7 - 15 nM, for E:T ratios of up to 1:50). In orthotopic xenograft models with the same set of cell lines, all three cell lines were efficiently eliminated by activated T cells in the presence of a single dose of FLT3 bispecific (dose range 10 to 100 μg/kg), confirming the anti-tumor activity of the bispecific antibody in vivo.The safety of Pfizer's FLT3 bispecific antibody was also studied in preclinical studies. We first analyzed gene expression of FLT3 and three other commonly considered AML targets (CD33, CD123, and CLL-1) in publicly available gene expression databases. FLT3 had the highest differential expression between AML and healthy tissues with blood and brain showing expression of low levels of FLT3 RNA. In tissue cross-reactivity studies, we detected little to no binding of the candidate FLT3 antibody to human brain tissue sections. In blood, the highest expression of FLT3 was in hematopoietic stem cells and progenitors, dendritic cells and monocytes consistent with previous publications and known roles of FLT3 in hematopoiesis and dendritic cell homeostasis. Of note, expression of FLT3 in monocytes and whole blood was the lowest compared to three other AML targets.To address any potential toxicities of FLT3 bispecific, we performed exploratory studies in cynomolgus monkeys. Affinity of FLT3 bispecific for cynomolgus FLT3 was ~20x lower than for human FLT3 (~1 nM), as determined in binding assays with cells expressing human and cynomolgus FLT3. No major clinical signs or toxicological findings were observed up to the highest dose level tested (3 mg/kg) following two administrations at weekly intervals. On target activity was demonstrated by nearly complete elimination of FLT3+ dendritic cells in the blood of treated monkeys two days after administration. In addition, we detected elimination of FLT3+ CD34+ stem cells in the bone marrow of treated monkeys two days after the second dose. Remarkably, both dendritic cells and FLT3+ CD34+ stem cells rebounded to baseline levels observed in control animals when analyzed at study termination, two weeks following the second dose. Consistent with minimal expression of FLT3 on healthy tissues and the presence of FLT3+ on rare blood subsets, cytokine increases typically associated with CD3 bispecific administration were minimal (only 6 to 17 and 46 to 89 fold over baseline for IFN-γ and IL-6, respectively, at the highest dose tested).In conclusion, the robust anti-tumor activity of Pfizer's FLT3 bispecific antibody combined with good tolerability in cynomolgus monkeys, reversible hematological toxicity and absence of non-hematological toxicity support its further clinical development in AML. DisclosuresDjuretic:Pfizer Inc.: Employment. Krishnamoorthy:Pfizer: Employment. Sommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Dettling:Maverick Therapeutics: Employment. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Chen:Pfizer: Employment. Hu:Pfizer: Employment. Given Chunyk:Pfizer: Employment. Lindquist:Pfizer: Employment. Potluri:Pfizer: Employment. Rickert:Pfizer: Employment. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Yeung:Pfizer: Employment, Patents & Royalties.

ALLO-819, an Allogeneic Flt3 CAR T Therapy Possessing an Off-Switch for the Treatment of Acute Myeloid Leukemia

▪Patients with relapsed acute myeloid leukemia (AML) have poor prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented clinical efficacy in hematological malignancies, leading to durable responses in heavily pretreated patients. Adoptive immunotherapies using T cells redirected against AML cells are being pursued as one option with potential curative intent. However, the development of autologous CAR T therapies presents a significant logistical and clinical challenge in a rapidly progressing disease setting such as AML due to the lag time of cell manufacturing. Additionally, harvesting sufficient numbers of healthy T cells from patients with AML may not always be possible. For these reasons the development of an off-the-shelf CAR T cell product may be of benefit. This work details the preclinical evaluation of ALLO-819, an allogeneic CAR T therapy targeting the receptor tyrosine kinase Flt3 (CD135), an AML target with high prevalence in all AML subtypes and limited expression outside of the hematopoietic tissue.To construct a Flt3 CAR, a panel of high affinity (KD values of 0.19 to 233 nM, determined at 37°C) fully-human antibodies was generated using phage display technology. Single-chain variable fragments (scFvs) recognizing different immunoglobulin domains of the extracellular region of Flt3 were inserted into second-generation CAR constructs and tested for their ability to redirect T cell specificity and effector function towards AML cells. A lead CAR exhibiting minimal tonic signaling and potent antitumor activity in orthotopic mouse models of AML (2.5x106 and 1x107 CAR T cells for Eol-1 and Molm-13, respectively) was selected for further engineering to incorporate a safety off-switch in cis. To accomplish this, short amino acid stretches mimicking epitopes for the FDA-approved antibody rituximab were inserted between the hinge and target-binding regions of the CAR. The CAR T cell phenotype and antitumor efficacy were not affected by the presence of the off-switch. In the presence of rituximab, Flt3 CAR T cells were efficiently lysed via complement-dependent cytotoxicity (~ 80 % CAR T cell depletion in 3 hours) in vitro and eliminated in peripheral blood and bone marrow of NSG mice (>100-fold and >300-fold, respectively).Allogeneic ALLO-819 Flt3 CAR T cells with a lower risk of TCR-mediated graft-versus-host disease and resistant to anti-CD52 antibody (alemtuzumab)-mediated lysis were generated by disruption of the T-cell receptor alpha chain (TRAC) and the CD52 loci using TALEN® gene-editing technology. Transient expression of TALEN® in Flt3 CAR T cells resulted in high-efficiency inactivation of both loci and had no impact on T cell phenotype or antitumor efficacy. ALLO-819 Flt3 CAR T cells co-cultured with primary AML blasts ex vivo displayed target-dependent activation, cytokine secretion and cytotoxic activity.Consistent with previous reports, we detected Flt3 expression on a subset of normal hematopoietic stem and progenitor cells (HSPCs) which also showed susceptibility to CAR T cell cytotoxicity. To evaluate off-tumor effects of Flt3 CAR T cells in vivo, NSG mice were administered T cells expressing a CAR with similar affinity to both mouse and human Flt3. Mouse-cross-reactive Flt3 CAR T cells exhibited off-tumor activity that was limited to a subset of bone marrow multipotent progenitors and correlated with antitumor efficacy. Administration of rituximab led to effective depletion of CAR T cells in peripheral blood that was followed by a rapid repopulation of HSPCs to levels observed in naïve mice.In summary, these results support the development of ALLO-819 Flt3 CAR T as a novel immunotherapy for the treatment of AML. DisclosuresSommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Nguyen:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Overexpression of functional human FLT3 ligand in Pichia pastoris

Growth factors and cytokines including FLT3 Ligand (FLT3L) have many applications in cellular and molecular biology studies, and also as therapeutical agents. FLT3L was expressed in Pichia pastoris through cloning from human leukemic K562 cell line as well as using a codon-optimized synthetic construct. Codon adaptation index (CAI) was increased from 55 in the native sequence up to 95 after optimization. Significant changes occurred in the codons for Pro, Arg, Leu and Ser toward the favored codons in P. pastoris. Both forms of expressed FLT3L (from the native and optimized sequences) were capable of stimulating proliferation of the FDC-P1 cells expressing human wild-type FLT3 (FD–FLT3–WT). Sequence optimization resulted in 55-fold increase in the yield of active FLT3L (290 μg/mL of product in the crude supernatant). Amino acid residues 27–162 are sufficient for the biological function of human FLT3L. Pichia pastoris is a highly efficient and cost-effective system for expression of endotoxin-free FLT3L; however, codon optimization is necessary for its optimal expression.

The human CD34(−)CD117(+)CD56(−)NKp44(−) population in secondary lymphoid tissue serves as a common progenitor to NK cells and ILC2s

Abstract The developmental pathways of human innate lymphoid cells (ILCs) have not been fully elucidated. We reported that human natural killer (NK) cells can derive from progenitor cells naturally residing in tonsils. We hypothesized that Group 2 ILCs (ILC2s) also develop in tonsils through a shared pathway. In support of this, ex vivo flow cytometry analyses of enriched human tonsil ILCs revealed a putative pathway of human CD294(+) ILC2 development stemming from a lineage (Lin)(−)CD34(−)CD117(+) population from which non-overlapping CD94(+) NK cells also emerged. Furthermore, when purified tonsil Lin(−)CD34(−)CD117(+) cells were cultured for 28 days with murine OP9 stroma expressing the human Notch ligand, Delta-like 1, in the presence of human interleukin-7 and Flt3 ligand, they produced mutually distinct and functionally mature populations of CD94(+) NK cells and CD294(+) ILC2s. The tonsil Lin(−)CD34(−)CD117(+) population is heterogeneous for CD56 and NKp44. Evaluation of the in vitro lineage differentiation potentials of subsets within the Lin(−)CD34(−)CD117(+) population according to NKp44 and CD56 revealed that NKp44(−)CD56(−) cells gave rise to both NK cells and ILC2s, whereas fractions expressing NKp44 and/or CD56 gave rise to NK cells but only to negligible ILC2s. In fact, the NKp44(−)CD56(−) population gave rise to a significantly higher percentage of ILC2s (7.50 ± 0.6) compared to the NKp44(+)CD56(+/−) populations (1.22 ± 0.4) (n = 5, p &amp;lt; 0.001). These data support a model whereby human NK cells and ILC2s can develop from a common Lin(−)CD34(−)CD117(+)CD56(−)NKp44(−) progenitor in tonsils and that the acquisition of NKp44 and/or CD56 restricts lineage differentiation potential towards the NK cell lineage.

A Phase II Randomized Study of CDX-1401, a Dendritic Cell Targeting NY-ESO-1 Vaccine, in Patients with Malignant Melanoma Pre-Treated with Recombinant CDX-301, a Recombinant Human Flt3 Ligand.

9589Background: Patients (pts) with high-risk melanoma have a 20-60% recurrence rate with 5-year OS between 45% and 70%. The adjuvant setting is an opportunity to test prevention vaccines that may have efficacy against disease recurrence. We evaluated CDX301, a recombinant human Flt3 ligand (Flt3L), plus vaccination with CDX1401, a fusion protein consisting of human monoclonal IgG1 antibody targeting the dendritic cell (DC) receptor DEC-205 linked to the NY-ESO-1 tumor antigen, in a phase II, open-label, multicenter, randomized study of subjects with resected melanoma, to determine whether immune responses to NY-ESO-1 elicited by vaccination with CDX1401 + poly-ICLC are substantially increased by prior expansion of circulating DC with Flt3L therapy. Methods: 60 pts with resected melanoma were randomized to two cohorts: Cohort 1 received CDX301 pretreatment (25 ug/kg SC x 10 days) in 2 of 4 monthly cycles of vaccination with CDX1401 (1mg IC) + poly-ICLC (2mg SC, days 1 and 2). Cohort 2 received 4 monthly c...

The Dual MEK/FLT3 Inhibitor E6201 Exerts Cytotoxic Activity against Acute Myeloid Leukemia Cells Harboring Resistance-Conferring FLT3 Mutations.

Fms-like tyrosine kinase 3 (FLT3) inhibition has elicited encouraging responses in acute myeloid leukemia (AML) therapy. Unfortunately, unless combined with a bone marrow transplant, disease relapse is frequent. In addition to the acquired point mutations in the FLT3 kinase domain that contribute to FLT3 inhibitor resistance, MEK/ERK signaling is persistently activated in AML cells even when FLT3 phosphorylation is continually suppressed. Thus, concomitant targeting of FLT3 and MAPK may potentially exert synergistic activity to counteract the resistance of AML cells to FLT3-targeted therapy. In this study, we investigated the antileukemia activity of a MEK1 and FLT3 dual inhibitor, E6201, in AML cells resistant to FLT3 inhibition. We found that E6201 exerted profound apoptogenic effects on AML cells harboring resistance-conferring FLT3 mutations. This activity appeared to be p53 dependent, and E6201-induced cytotoxicity was retained under hypoxic culture conditions and during coculture with mesenchymal stem cells that mimic the AML microenvironment. Furthermore, E6201 markedly reduced leukemia burden and improved the survival of mice in a human FLT3-mutated AML model. Collectively, our data provide a preclinical basis for the clinical evaluation of E6201 in AML patients harboring FLT3 mutations, including those who relapse following FLT3-targeted monotherapy.

Differential Inhibition of FLT3-ITD, FLT3-WT, and KIT By Quizartinib As Assessed By Modified PIA Assay

The plasma inhibitory activity (PIA) assay has been used to determine if sufficient exposure of FLT3 kinase inhibitors have been achieved to inhibit their target following clinical administration. Traditionally, clinical plasma samples have been used undiluted to determine inhibitory activity. In the case of quizartinib (AC220), undiluted plasma often results in full inhibition of ex vivo FLT3 activity, regardless of dose administered. To more closely examine the inhibitory properties of circulating quizartinib in a clinical trial, we modified the western blot PIA assay to utilize MSD-based phospho (p)FLT3 and pKIT ELISAs. This modification allows for the comparison of inhibitory activity between samples, resolution of activity against multiple targets from a single subject sample, as well as demonstrates the differences between administered quizartinib doses.A total of 76 relapsed/refractory FLT3-ITD AML subjects were enrolled in a Phase 2b study. Of those, 56 subjects (30 at 30 mg/day; 26 at 60 mg/day) had sufficient pre-dose and either day 8 or 15 plasma samples available for evaluation. Samples were tested undiluted and serially 4-fold diluted into normal human plasma. FLT3 inhibitory activity was determined in Ba/F3 cells exogenously expressing human FLT3 with an internal tandem duplication (ITD) mutation (ITD cells), and in THP-1 cells, where wild-type (WT) FLT3 phosphorylation was FLT3 ligand induced. Inhibitory activity on c-KIT in H256 cells with SCF-induced phosphorylation was also determined. Percent inhibition was calculated by comparing post-dose samples to equivalently diluted pre-dose samples.As in previous western blot-based PIA assays, undiluted plasma samples contained sufficient quizartinib and its active metabolites to inhibit FLT3 phosphorylation in ITD cells by 98.0 ± 3.3% and 100.4 ± 4.0% at 30 and 60 mg doses, respectively, suggesting exposures that completely inhibit cellular FLT3-ITD activity. Diluting the plasma samples allowed for determination of the IT50 (the plasma dilution titer resulting in 50% inhibition of kinase activity) to assess the excess active drug in each sample. For subjects receiving 30 mg, the median IT50 was 21.0 (range 7.3-45.9) (inverse dilution). For subjects receiving 60 mg, the median IT50 was 44.8 (range 19.1-135.5), indicating the expected 2-fold difference in inhibitory activity. The percent inhibition levels were significantly different between 30 and 60 mg doses at all dilutions in the ITD cells. For FLT3-WT in THP-1 cells, the mean inhibitions of ligand-induced phosphorylation in the undiluted samples were 77.6 ± 9.6% and 90.1 ± 5.6% with the two doses, indicating that FLT3-WT is not as well inhibited by quizartinib. The median IT50s were 4.3 (range 3.0-8.0) and 6.6 (range 3.7-13.6) for the 30 and 60 mg doses, consistent with less excess drug relative to FLT3-WT inhibition. KIT inhibitory activity was weaker at these doses of quizartinib. Undiluted plasmas had a mean inhibition of 43.3 ± 16.1% and 63.9 ± 14.1% at 30 and 60 mg, so IT50s could only be calculated for one subject at 30 mg and 10 subjects at 60 mg. KIT inhibition levels were still significantly different between the two doses in the undiluted and 1:4 diluted plasma samples, but were largely eliminated by the 1:16 dilution.Measured plasma levels of quizartinib and its metabolite AC886 were used to calculate the plasma IC50 values for each cell line. For ITD cells, the median IC50 was 20.5 nM (range 6.9-64.6 nM). In the THP-1 cells, it was 129 nM (range 52.1-552 nM). For KIT inhibition, the median IC50 was 442 nM (range 167-1577 nM). Of note, the median quizartinib + AC886 levels were 370 nM (range 205-802 nM) for 30 mg subjects, and 917 nM (range 291-4234 nM) for 60 mg subjects, indicating on average, the 30 mg subjects were 18-fold above the the IC50 for FLT3-ITD inhibition, and the 60 mg subjects were 45-fold above this IC50, and drug levels were 3-fold and 7-fold above the IC50 for FLT3-WT in the two doses. For KIT inhibition, most 30 mg subjects had drug levels below the KIT IC50, while 60 mg subjects had drug levels only 2-fold above it.These results suggest that quizartinib, when dosed at 30 mg or 60 mg achieved peripheral exposures sufficient for complete inhibition of FLT3-ITD as assessed by the PIA assay. At these same doses the degree of inhibition of FLT3-WT, and especially KIT is reduced. Implications of this data for patient response and myelosuppression will be available for presentation. DisclosuresNepomuceno:Ambit Biosciences Corporation: Employment. Rooks:Ambit Biosciences Corporation: Employment. Gardner:Ambit Biosciences Corporation: Employment. Armstrong:Ambit Biosciences Corporation: Employment, Equity Ownership.

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

Establishment of hematological malignancy model in ex vivo cell culture

The aim of this study was to develop an ex vivo cell culture system for establishing the hematological malignancy model. Mouse bone marrow cells were transfected with GFP-expressed retroviral vectors encoding various leukemia/lymphoma-associated fusion proteins (TEL-PDGFR, Rabaptin5-PDGFR, p210BCR-ABL, AML1-ETO, NPM-ALK). After transfection, the cells were cultured in IMDM containing 10% FCS without growth factors, or with one of the following growth factor combinations: (1) murine c-kit ligand (KL) plus human flt3 ligand (FL); (2) IL-3, thrombopoietin, G-CSF, and hyper-IL-6 (3/T/G/H6); (3) KL/FL plus 3/T/G/H6. The flow cytometry was used to detect the ability of combinations of growth factors to complement the oncogene fusion protein to support self-renewal of the transfected cells. The results showed that the transfected cells could be amplified sustainably in the logarithmic growth way. The indicated combination of c-Kit ligand (KL) with flt-3 ligand (FL) supported the self-renewal of the marrow cells transfected with vectors encoding TEL-PDGFR, Rabaptin5-PDGFR, AML1-ETO and NPM-ALK, in addition to KL/FL, the self-renewal of p210 BCR-ABL transfected-marrow cells also required IL-3. The morphology of cells emerged from culture can be the predictor of the corresponding oncogene-associated malignancy. It is concluded that this study establishes a culture system ex vivo which provides a generalized method for studying hematological malignancies, and may facilitate the screening for therapeutic agents.