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. 2015 Dec;64(12):1575-86.
doi: 10.1007/s00262-015-1763-8. Epub 2015 Oct 15.

Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo

Christian Cremer  1 , Hanna Braun  1 , Radoslav Mladenov  2 , Lea Schenke  1 , Xiaojing Cong  3   4 , Edgar Jost  5 , Tim H Brümmendorf  5 , Rainer Fischer  2   6 , Paolo Carloni  3   4 , Stefan Barth  1   7   8 , Thomas Nachreiner  9
Affiliations

Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo

Christian Cremer et al. Cancer Immunol Immunother. 2015 Dec.

Abstract

Immunotoxins are fusion proteins that combine a targeting component such as an antibody fragment or ligand with a cytotoxic effector component that induces apoptosis in specific cell populations displaying the corresponding antigen or receptor. Human cytolytic fusion proteins (hCFPs) are less immunogenic than conventional immunotoxins because they contain human pro-apoptotic enzymes as effectors. However, one drawback of hCFPs is that target cells can protect themselves by expressing endogenous inhibitor proteins. Inhibitor-resistant enzyme mutants that maintain their cytotoxic activity are therefore promising effector domain candidates. We recently developed potent variants of the human ribonuclease angiogenin (Ang) that were either more active than the wild-type enzyme or less susceptible to inhibition because of their lower affinity for the ribonuclease inhibitor RNH1. However, combining the mutations was unsuccessful because although the enzyme retained its higher activity, its susceptibility to RNH1 reverted to wild-type levels. We therefore used molecular dynamic simulations to determine, at the atomic level, why the affinity for RNH1 reverted, and we developed strategies based on the introduction of further mutations to once again reduce the affinity of Ang for RNH1 while retaining its enhanced activity. We were able to generate a novel Ang variant with remarkable in vitro cytotoxicity against HL-60 cells and pro-inflammatory macrophages. We also demonstrated the pro-apoptotic potential of Ang-based hCFPs on cells freshly isolated from leukemia patients.

Keywords: Angiogenin; Human cytolytic fusion protein; Leukemia; RNH1; Site-directed mutagenesis; Targeted therapy.

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Conflict of interest statement

The authors have no conflict of interest to disclose.

Figures

Fig. 1
Fig. 1
Representative yeast tRNA cleavage assay. The hCFP-mediated degradation of tRNA was demonstrated by incubating 1–0.25 µg of each fusion protein with 600 ng yeast tRNA for 90 min at 37 °C to measure the dose-dependent degradation efficiency. We used 100 ng RNase A as a positive control (+) and tRNA in RNase-free reaction buffer as a negative control (−). The degradation efficiency in the presence of RNH1 was tested in a representative experiment by adding 5 U commercial RNH1 to 1 µg hCFP under the same cleavage conditions. H22-Ang GGRR/QG/DHmut achieved the highest cleavage efficiency in the presence of RNH1
Fig. 2
Fig. 2
Relative enzymatic activities of hCFPs. The hCFPs were incubated with RNaseAlert™ overnight at room temperature, and the fluorescence emission was normalized against the fluorescence background (0 %) and the maximal fluorescence of wild-type H22-Ang (100 %). H22-SNAP was used as a negative control under the same conditions. Relative enzymatic activities are expressed as means of three independent experiments ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t test (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001)
Fig. 3
Fig. 3
Susceptibility of hCFPs to inhibition by RNH1. We incubated 100 nM of each hCFP with 50 nM of RNaseAlert™ and 2–200 nM RNH1 overnight at room temperature, and the fluorescence emission was normalized against the fluorescence background (0 %) and the desired maximum fluorescence intensities without inhibition (100 %). Individual inhibition profiles were obtained by plotting the logarithmic protein concentration of RNH1 against the relative enzymatic activities. The sigmoidal dose-dependent inhibition was fitted using a log (inhibitor) versus response fit. Values are expressed as mean ± SEM of three independent experiments
Fig. 4
Fig. 4
In vitro and ex vivo binding activities of hCFPs. We incubated 1 µg hCFP with hIFNγ-stimulated HL-60, hM1Φ and L-540cy cells (a) or leukemia-derived PBMCs (b) for 30 min on ice. An Alexa Fluor 488 detection antibody was added to the samples for 20 min on ice in the dark, and binding was demonstrated by flow cytometry. A fluorescence shift compared to the detection antibody control indicated binding. The HL-60, L–540cy and hM1Φ target cell populations were gated as indicated (a, SSC-H/FSC-H), whereas all PBMCs were analyzed for specific binding
Fig. 5
Fig. 5
Cytotoxicity of hCFPs against HL-60 and hM1Φ cells. Stimulated HL-60 and hM1Φ cells (a) were used to evaluate hCFP cytotoxicity. The cells were exposed to serial dilutions of hCFPs for 72 h and treated with either zeocin or PBS as controls. Viable cells were detected using a XTT colorimetric assay. Target cell specificity was demonstrated by the absence of cytotoxicity against CD64− L-540cy cells. EC50 values were calculated after sigmoidal dose–response fitting (b). EC50 values are expressed as mean ± SD from at least three independent experiments. Statistical comparisons were carried out using a two-tailed unpaired Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)
Fig. 6
Fig. 6
Representative ex vivo depletion of isolated leukemia PBMCs. PBMCs were isolated by density gradient centrifugation from the peripheral blood (a–c) or bone marrow (d) of acute or chronic myelomonocytic leukemia patients. The cells were cultivated in the presence of 200 nM hCFPs and controls for 12 h at 37 °C in a 5 % CO2 atmosphere. The cells were then double-stained with annexin V-eGFP and PI before analysis by flow cytometry. The sum of early-apoptotic and late-apoptotic cells is shown as the mean of duplicates ± SD in comparison with the controls
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