Diphtheria toxin fused to variant interleukin-3 provides enhanced binding to the interleukin-3 receptor and more potent leukemia cell cytotoxicity

Liu, Tie Fu; Urieto, Jeffrey O; Moore, Joseph Earle; Miller, Mark Steven; Lowe, Albert; Thorburn, Andrew; Frankel, Arthur E.

doi:10.1016/j.exphem.2003.11.010

Cited by 27 publications

(10 citation statements)

References 29 publications

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“…Furthermore, researchers also found that the variant DT388IL3 (△125-133) showed an enhanced binding to the human IL3R and greater cytotoxicity to human AML cell lines and LSCs than the wild-type DT388IL3 [ 42 ]. Following the same way, our laboratory constructed and expressed the fusion protein antiCD3Fv-IL3 and discovered the fusion protein was unstable and could easily break in the end of the C-terminal (data not shown), which was also consistent with some combinatorial mutagenesis studies [ 22 - 25 ]. So we engineered the fusion protein antiCD3Fv-⊿IL3 (⊿125-133) and its disulfide-stabilized antiCD3Fv-⊿IL3, which combined the advantages of both the specificity of humoral immunity and the efficiency of cellular immunity compared with fusion proteins DT388IL3.…”

Section: Discussionsupporting

confidence: 77%

“…The ligand-receptor-binding activity is considered to be very potent. To further increase the stability of the ligand-receptor binding, combinatorial mutagenesis studies by several laboratories showed that deletion of eight C-terminal amino acid residues from IL3 (⊿125-133) or the variant K116W resulted in even higher affinity interactions with IL3R and greater cytotoxicity against human leukemic stem cells [ 22 - 25 ].…”

Section: Introductionmentioning

confidence: 99%

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AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells

Fan

Zhang

et al. 2015

J Hematol Oncol

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BackgroundLeukemic stem cells (LSCs) are frequently seen as a cause of treatment failure and relapse in patients with acute myeloid leukemia (AML). Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs. The identification of targets on the cell surface of LSCs is getting more and more attention. Among these, CD123, also known as the interleukin-3 (IL3)-receptor α chain, has been identified as a potential immunotherapeutic target due to its overexpression on LSCs in AML as well as on AML blasts, rather than normal hematopoietic stem cells.MethodsWe constructed a CD123-targeted fusion protein antiCD3Fv-⊿IL3, with one binding site for T cell antigen receptor (TCRCD3) and the other for CD123, by recombinant gene-engineering technology. Cysteine residues were introduced into the V domains of the antiCD3Fv segment to enhance its stability by locking the two chains of Fv together with disulfide covalent bonds. The stability and cytotoxicity of the two fusion proteins were detected in vitro and in vivo.ResultsBoth fusion proteins were produced and purified from Escherichia coli 16C9 cells with excellent yields in fully active forms. High-binding capability was observed between these two fusion proteins and human IL3R, leading to the specific lysis of CD123-expressing cell lines KG1a; also, mononuclear cells from primary AML patients were inhibited in a colony forming assay in vitro, presumably by redirecting T lymphocytes in vitro. In addition, they displayed an antileukemic activity against KG1a xenografts in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, especially disulfide-stabilized (ds)-antiCD3Fv-⊿IL3 for its improved stability.ConclusionsThese results suggest that both fusion proteins display the antileukemic activity against CD123-expressing cell lines as well as leukemic progenitors in vitro and in vivo, especially ds-antiCD3Fv-⊿IL3. They could be the promising candidates for future immunotherapy of AML.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0109-5) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 77%

Section: Introductionmentioning

confidence: 99%

AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells

Fan

Zhang

et al. 2015

J Hematol Oncol

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“…The IL-3 variants were designed based on a previous study that IL-3 [K116W] and IL-3 [Δ125-133] enhanced binding and cytotoxicity of diphtheria toxin-IL3 fusion proteins to leukemia cells. 33 Western blot analysis was performed to verify the production of these proteins. As shown in Figure 1b, the presence of the 6-his-tag, IL-3 and sCAR was detected.…”

Section: Resultsmentioning

confidence: 99%

CD123 targeting oncolytic adenoviruses suppress acute myeloid leukemia cell proliferation in vitro and in vivo

et al. 2014

Blood Cancer Journal

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We report here a novel strategy to redirect oncolytic adenoviruses to CD123 by carry a soluble coxsackie-adenovirus receptor (sCAR)-IL3 expression cassette in the viral genome to form Ad.IL3, which sustainably infected acute myeloid leukemia (AML) cells through CD123. Ad.IL3 was further engineered to harbor gene encoding manganese superoxide dismutase (MnSOD) or mannose-binding plant lectin Pinellia pedatisecta agglutinin (PPA), forming Ad.IL3-MnSOD and Ad.IL3-PPA. As compared with Ad.IL3 or Ad.sp-E1A control, Ad.IL3-MnSOD and Ad.IL3-PPA significantly suppressed in vitro proliferation of HL60 and KG-1 cells. Elevated apoptosis was detected in HL60 and KG-1 cells treated with either Ad.IL3-MnSOD or Ad.IL3-PPA. The caspase-9–caspase-7 pathway was determined to be activated by Ad.IL3-MnSOD as well as by Ad.IL3-PPA in HL60 cells. In an HL60/Luc xenograft nonobese diabetic/severe-combined immunodeficiency mice model, Ad.IL3-MnSOD and Ad.IL3-PPA suppressed cancer cell growth as compared with Ad.IL3. A significant difference of cancer cell burden was detected between Ad.IL3 and Ad.IL3-PPA groups at day 9 after treatment. Furthermore, Ad.IL3-MnSOD significantly prolonged mouse survival as compared with Ad.sp-E1A. These findings demonstrated that Ad.IL3-gene could serve as a novel agent for AML therapy. Harboring sCAR-ligand expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting serotype 5 adenovirus infection.

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“…The relatively high expression of IL3Rα in a number of leukaemias has presented a window of opportunity to develop new therapeutic approaches directed to IL3Rα such as monoclonal antibodies (MAbs) 8 , 31 , 32 , T cells engineered to express chimeric antigen receptors (CAR T cells) 33 and toxin conjugates with IL-3 34 – 37 . The latter was originally only partially successful as half the samples proved resistant to the fusion protein 38 . The potency of the toxin-IL-3 fusion protein was improved dramatically by introducing the K116W substitution into the IL-3 portion 38 – 40 .…”

Section: Discussionmentioning

confidence: 99%

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling

et al. 2018

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The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.

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Diphtheria toxin fused to variant interleukin-3 provides enhanced binding to the interleukin-3 receptor and more potent leukemia cell cytotoxicity

Cited by 27 publications

References 29 publications

AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells

AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells

CD123 targeting oncolytic adenoviruses suppress acute myeloid leukemia cell proliferation in vitro and in vivo

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling

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