Human Flt3 Is Expressed at the Hematopoietic Stem Cell and the Granulocyte/Macrophage Progenitor Stages to Maintain Cell Survival

Kikushige, Yoshikane; Yoshimoto, Goichi; Miyamoto, Toshihiro; Iino, Tadafumi; Mori, Yasuo; Iwasaki, Hiromi; Niiro, Hiroaki; Takenaka, Katsuto; Nagafuji, Koji; Harada, Mine; Ishikawa, Fumihiko; Akashi, Koichi

doi:10.4049/jimmunol.180.11.7358

Cited by 137 publications

(118 citation statements)

References 58 publications

(64 reference statements)

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“…9 More recently, humanized antibodies against more specific antigens on leukemic cells, such as the FMS-related tyrosine kinase 3 (FLT3, CD135; clinical trials ID NCT00887926) and the a-chain of the interleukin-3 receptor (CD123), 10 have been developed to the stage of early clinical trials. These antibodies, however, have not been optimized by recombinant antibody technology, and the antigens recognized are known to be expressed on dendritic cells (DCs), normal hematopoietic stem cells, 11 and ---in the case of CD123---cultured mast cells 12 and basophils, 13,14 raising concerns about potential toxicity towards these cells.…”

Section: Introductionmentioning

confidence: 99%

Generation, selection and preclinical characterization of an Fc-optimized FLT3 antibody for the treatment of myeloid leukemia

et al. 2012

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The therapeutic efficacy of humanized or chimeric second-generation antitumor antibodies is clearly established, but often limited. In recent years, defined modifications of the glycosylation pattern or the amino-acid sequence of the human immunoglobulin G1 Fc part have resulted in the development of third-generation antibodies with improved capability to recruit Fc receptor-bearing effector cells. The first antibodies of this kind, currently evaluated in early clinical trials, are directed against lymphoma-associated antigens. Fc-engineered antibodies targeting myeloid leukemia are not yet available. We here report on the generation and preclinical characterization of an Fc-optimized antibody directed to the FMS-related tyrosine kinase 3 (FLT3), an antigen expressed on the leukemic blasts of all investigated patients with acute myeloid leukemia (AML). This antibody, termed 4G8SDIEM, mediated markedly enhanced cellular cytotoxicity against FLT3-expressing cell lines as well as blasts of AML patients. FLT3 expression levels on AML cells varied between 300 and 4600 molecules/cell and, in most cases, were substantially higher than those detected on normal hematopoietic precursor cells and dendritic cells (approximately 300 molecules/cell). Antibody-mediated cytotoxicity against these normal cells was not detectable. 4G8SDIEM has been produced in pharmaceutical quality in a university-owned production unit and is currently used for the treatment of leukemia patients.

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Section: Introductionmentioning

confidence: 99%

Generation, selection and preclinical characterization of an Fc-optimized FLT3 antibody for the treatment of myeloid leukemia

et al. 2012

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“…Recent studies on the FLT3 expression have indicated distinct differences between the human and murine hematopoietic systems. While the expression of murine FLT3 is restricted to multipotent and lymphoid progenitor cells incapable of self-renewal, the human FLT3 is expressed on primitive hematopoietic stem cells and on progenitors along both the lymphoid and the granulocyte/macrophage pathway [8]. The main functions of FLT3 include induction of proliferation, differentiation and survival of normal hematopoietic progenitor cells.…”

Section: Introductionmentioning

confidence: 99%

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

Razumovskaya¹,

Masson²,

Khan

et al. 2009

Experimental Hematology

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Objective. Fms-like tyrosine kinase-3 (FLT3), a growth factor receptor normally expressed in hematopoietic progenitor cells, has been shown to have an important role in the development of acute myeloid leukemia c due to activating mutations. FLT3 mutations are found in approximately one third of AML patients and correlate with a poor prognosis, thus making the FLT3 receptor a potential therapeutic target. The aim of the investigation is to analyze the kinetics and specificity of FLT3 autophosphorylation in wild-type FLT3 as well as in the oncogenic FLT3 mutants.Methods. We have used Ba/F3 cells stably expressing either wild-type, ITD or D835Y mutants of FLT3 in order to compare the site selectivity of tyrosine phosphorylation sites. By the use of a panel of phosphospecific antibodies directed against potential tyrosine phosphorylation sites in FLT3, we identified several novel phosphorylation sites in FLT3 and studied the kinetics and specificity of ligand-induced phosphorylation in living cells.Results. Eight phosphorylated tyrosines (pY589, pY591, pY599, pY726, pY768, pY793, pY842 and pY955) were investigated and shown to be differentially phosphorylated in the wild-type versus the mutated receptors. Furthermore, we show that tyrosines 726, 793 and 842 are novel phosphorylation sites of FLT3 in intact cells. Conclusion.In this study, we have looked at the site-specific phosphorylation in the wild-type FLT3 in comparison to the mutants found in AML. We observed not only quantitative changes but more importantly, qualitative differences in the phosphorylation patterns of the wild-type and the mutated FLT3 receptors, which might contribute to the understanding of the mechanisms by which FLT3 contributes to AML in patients with mutations in FLT3.

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“…12,13 Several downstream progenitors with myeloid and/or lymphoid potential continue to express FLT3 whereas megakaryocyte/erythrocyte progenitors do not. 11,[14][15][16][17][18] With the exception of dendritic cells (DC), which retain FLT3 on their surface, FLT3 expression is downregulated as cells undergo myeloid or lymphoid commitment. Deletion of either FLT3 or FLT3L results in defects in the developmental potential of myeloid/lymphoid progenitors underscoring the importance of FLT3 in their development.…”

Section: Introductionmentioning

confidence: 99%

In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development

et al. 2014

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© F e r r a t a S t o r t i F o u n d a t i o nshown to be crucial in sustaining adult B-cell lymphopoiesis. 21 However, ablation of the FLT3/FLT3L axis alone did not result in a complete block in the generation of any hematopoietic lineage, suggesting that FLT3L might exert its crucial role in hematopoiesis through interactions with other cytokines, such as interleukin-7 (IL-7) or stem cell factor. 19,22,23 To elucidate the specific action of FLT3L on hematopoiesis in vivo, FLT3L has been administered, with the results confirming the important role of FLT3L in DC generation. 24 We have previously shown that apart from DC, FLT3L injection leads to transient expansion of a FLT3 + progenitor population with lymphoid and myeloid potential. 25 In order to evaluate the role of FLT3L in the development of different hematopoietic lineages, in this study we investigated the effects of sustained over-expression of FLT3L in a transgenic mouse model. Our study confirms the positive role of FLTL in DC development and highlights the importance of this cytokine in the survival and expansion of lymphoid and myeloid progenitors. Furthermore, our data provide evidence for an instructive role of FLT3L in hematopoietic development. Methods MiceAll mice used in this study were bred and maintained in our animal facility under pathogen-free conditions and all animal experiments were performed in accordance with institutional guidelines (permission numbers 1887 and 1888). Immunization to induce a Tdependent antibody response and FLT3L treatment of mice were carried out as previously described. 25 Cell culturesST2, OP9 and OP9 stromal cells expressing Notch ligand Deltalike 1 (OP9DL1) were maintained in IMDM supplemented with 5 x 10 -5 M β-mercaptoethanol, 1 mM glutamine, 0.03% w/v Primatone (Quest Naarden, the Netherlands), 100 U/mL penicillin, 100 μg/mL streptomycin and 5% fetal bovine serum. Co-cultures of stromal cells with sorted progenitor cells were performed as previously described 25 (and Online Supplementary Materials and Methods). Platelet countsBlood was drawn from the tail vein of mice and incubated with 1% ammonium oxalate for 10 min at room temperature. Following incubation, live cells were counted in a Neubauer hemocytometer. ImmunofluorescenceSpleens were snap-frozen and embedded in OCT-compound (Sakura, Zoetermeer, the Netherlands), and 5 μm sections were prepared. Sections were fixed in acetone for 10 min, air-dried for 60 min and subsequently stained with fluorescein isothiocyanatelabeled anti-CD90, phycoerythrin-labeled anti-IgM and allophycocyanin-labeled anti-CD11c antibodies for 30 min. Results Splenomegaly and lymphadenopathy in FLT3L transgenic miceTo investigate the effect of prolonged FLT3L overexpression, we generated mice expressing the human FLT3L gene under the control of the β-actin promoter (hereafter referred to as FLT3L-Tg mice). FLT3L levels in the blood were in the range of 500-1000 ng/mL, as assesed by enzyme-linked immunosorbent assay using an antihuman FLT3L antibody developed in our ...

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Human Flt3 Is Expressed at the Hematopoietic Stem Cell and the Granulocyte/Macrophage Progenitor Stages to Maintain Cell Survival

Cited by 137 publications

References 58 publications

Generation, selection and preclinical characterization of an Fc-optimized FLT3 antibody for the treatment of myeloid leukemia

Generation, selection and preclinical characterization of an Fc-optimized FLT3 antibody for the treatment of myeloid leukemia

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development

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