Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugate - PubMed (original) (raw)
doi: 10.2119/molmed.2015.00210. Epub 2016 Jul 22.
Connie Duong 1 2, Sakiko Yoshida 1 2, Michael Oestergaard 3, Cathy Chen 1 2, Rachael Peralta 3, Shuling Guo 3, Punit P Seth 3, Yueju Li 4, Laurel Beckett 4, Jong Chung 1, Jan Nolta 2, Nitin Nitin 5, Joseph M Tuscano 6
Affiliations
- PMID: 27455414
- PMCID: PMC5082301
- DOI: 10.2119/molmed.2015.00210
Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugate
Noriko Satake et al. Mol Med. 2016 Oct.
Abstract
The exponential rise in molecular and genomic data has generated a vast array of therapeutic targets. Oligonucleotide-based technologies to down regulate these molecular targets have promising therapeutic efficacy. However, there is relatively limited success in translating this into effective in vivo cancer therapeutics. The primary challenge is the lack of effective cancer cell-targeted delivery methods, particularly for a systemic disease such as leukemia. We developed a novel leukemia-targeting compound composed of a monoclonal antibody directly conjugated to an antisense oligonucleotide (ASO). Our compound uses an ASO that specifically targets the transcription factor MAX dimerization protein 3 (MXD3), which was previously identified to be critical for precursor B cell (preB) acute lymphoblastic leukemia (ALL) cell survival. The MXD3 ASO was conjugated to an anti-CD22 antibody (αCD22 Ab) that specifically targets most preB ALL. We demonstrated that the αCD22 Ab-ASO conjugate treatment showed MXD3 protein knockdown and leukemia cell apoptosis in vitro. We also demonstrated that the conjugate treatment showed cytotoxicity in normal B cells, but not in other hematopoietic cells, including hematopoietic stem cells. Furthermore, the conjugate treatment at the lowest dose tested (0.2mg/kg Ab for 6 doses - twice a week for 3 weeks) more than doubled the mouse survival time in both Reh (median survival time 20.5 vs. 42.5 days, p<0.001) and primary preB ALL (median survival time 29.3 vs. 63 days, p<0.001) xenograft models. Our conjugate that uses αCD22 Ab to target the novel molecule MXD3, which is highly expressed in preB ALL cells, appears to be a promising novel therapeutic approach.
Keywords: biochemistry; cell biology; gene expression; molecular biology; oncology; pediatrics.
Conflict of interest statement
DISCLOSURE
MO, SG, PPS and RP are employees and stockholders of Ionis Pharmaceuticals. This work was supported by research funding from The Hartwell Foundation, Keaton Raphael Memorial Foundation, National Center for Advancing Translational Sciences, NIH, through grant #UL1 TR000002, and CTSC-MCRTP (Satake) and the California Institute for Regenerative Medicine and NIH Transformative grant R01GM099688 (Nolta). Statistical support was provided through the Biostatistics Shared Resource, UC Davis Comprehensive Cancer Center Support Grant P30CA093373.
Figures
Figure 1.
αCD22 Ab-MXD3 ASO conjugate demonstrates MXD3 knockdown leading to cell apoptosis in preB ALL cells. (A) Reh cells treated with αCD22 Ab-MXD3 or αCD22 Ab-control ASO conjugate, or left untreated, were measured for MXD3 protein expression by fluorescent immunocytochemistry 4 h after treatment. The concentration of the conjugate in each well was 0.5 umol/L. Images were acquired at 40× magnification/1.4 numerical aperture at room temperature using a Nikon Ti-U inverted microscope and NIS-Elements BR software. Scale bar indicates 50 μm. The overlay pictures show a composite image of both MXD3 protein (with Alexa488) and nuclei (with DAPI). The images shown are from one representative experiment out of three experiments. (B) Mean fluorescence intensity (MFI) was used to quantify MXD3 protein expression. Each bar represents the average MFI of all measured cells per treatment type from three independent experiments. MXD3 αCD22 Ab conjugate versus untreated (**p = 0.008) versus αCD22 Ab-control ASO conjugate (*p = 0.032). (C) Cell apoptosis measured by annexin V using flow cytometry. Dot plots shown are from one representative experiment out of three experiments. (D) Quantification of annexin V positive cells. Data are means from three independent experiments. Error bars represent SEM (n = 3 for each time point). The cells treated with αCD22 Ab-MXD3 ASO conjugate showed significantly more annexin V positive cells than either the untreated cells (***p < 0.001) or those treated with the αCD22 Ab-control ASO conjugate (***p < 0.001), at both 2 and 4 h. (E) Cell apoptosis measured by caspase 3 and 7. Cells were treated the same way as in (D) with the same control. Histograms represent caspase measured by luminescence signal at 2 and 4 h after treatment. Data are an average of three independent experiments in triplicate. Error bars represent SEM (n = 9 for each time point). Cells treated with the αCD22 Ab-MXD3 ASO conjugate showed significantly higher average caspase than either the untreated cells (***p < 0.001) or those treated with the αCD22 Ab-control ASO conjugate (***p < 0.001), at both 2 and 4 h.
Figure 2.
αCD22 Ab-MXD3 ASO conjugate has cytotoxicity in normal B cells, but not in HSCs. (A) Normal B cells express low levels of MXD3 and treatment with the αCD22 Ab-MXD3 ASO conjugate showed knockdown. Images were acquired at 40× magnification/1.4 numerical aperture at room temperature using a Nikon Ti-U inverted microscope and NIS-Elements BR software. Scale bar indicates 50 μm. Free αCD22 Ab (azide-conjugated) + free MXD3 ASO also showed low levels of non-specific knockdown. The images shown are from one representative experiment out of three experiments. (B) MXD3 protein knockdown quantified using MFI. Each bar represents the average MFI of all measured cells per treatment type from three independent experiments. (C) Accelerated cell death in B cells treated with the αCD22 Ab-MXD3 ASO conjugate. Data points indicate mean values of independent cell counts in triplicate from three independent experiments in triplicate. Data as mean ± SEM (n = 9). The MXD3 αCD22 Ab-ASO conjugate versus free αCD22 Ab (azide-conjugated) + free MXD3 ASO (***p < 0.001).
Figure 3.
αCD22 Ab-MXD3 ASO conjugate shows significant in vivo dose-dependent therapeutic efficacy in Reh human leukemia mouse model. Kaplan-Meier survival curve for mice inoculated with Reh cells and treated with the αCD22 Ab-MXD3 ASO conjugate. Data from two independent experiments were combined (n = 4 or 8). In the first experiment, the mice were treated with PBS, free αCD22 Ab (1 mg/kg) plus free MXD3 ASO (0.8 mg/kg), and two different doses of the αCD22 Ab-MXD3 ASO conjugate (0.2 mg/kg or 1 mg/kg of the Ab). In the second experiment, the mice were treated with free αCD22 Ab (1, 5 or 10 mg/kg) plus free MXD3 ASO (0.8, 4 or 8 mg/kg), respectively, and three different doses of the αCD22 Ab-MXD3 ASO conjugate (1, 5 or 10 mg/kg of the Ab). PBS versus conjugate at any dose (0.2, 1, 5 or 10 mg/kg of the Ab) (**p < 0.01, ***p < 0.001, **p < 0.01 or **p < 0.01, respectively). Free αCD22 Ab (1 mg/kg) plus free MXD3 ASO (0.8 mg/kg) versus conjugate at any dose (0.2, 1, 5 or 10 mg/kg of the Ab) (**p < 0.01, ***p < 0.001, **p < 0.01 or **p < 0.01, respectively). Free Ab plus free ASO versus conjugate at the equivalent dose of the Ab (1, 5 or 10 mg/kg) (***p < 0.001, *p = 0.01 or **p < 0.01, respectively).
Figure 4.
αCD22 Ab-MXD3 ASO conjugate shows in vivo anti-leukemic effects against engrafted leukemia cells in mice. (A) The αCD22 Ab-MXD3 ASO conjugate treatment showed MXD3 knockdown in the engrafted Reh cells at 8 h after treatment. MXD3 protein expression was measured in the cells harvested from BM. Images were acquired at 40× magnification/1.4 numerical aperture at room temperature using a Nikon Ti-U inverted microscope and NIS-Elements BR software. Scale bar indicates 50 μm. (B) The αCD22 Ab-MXD3 ASO conjugate treatment induced cell apoptosis in the engrafted Reh cells at 8 and 24 h after treatment. Cells were stained for human CD22 and annexin V. Red dots (lower quadrants): murine cells (CD22 negative), purple dots (upper left quadrant): live Reh cells (CD22 positive annexin V negative), and blue dots (upper right quadrant): apoptotic Reh cells (CD22 and annexin V positive) in BM.
Figure 5.
αCD22 Ab-MXD3 ASO conjugate shows significant in vivo therapeutic efficacy in two human patient-derived leukemia mouse models. Kaplan-Meier survival curves for mice inoculated with two patient-derived leukemia cells (A and B). The mice were treated with PBS, free αCD22 Ab (1 mg/kg) plus free MXD3 ASO (0.8 mg/kg), and two different doses of the αCD22 Ab-MXD3 ASO conjugate (0.2 mg/kg or 1 mg/kg of the Ab). (A) PBS versus conjugate at both doses (***p < 0.001). Conjugate 0.2 mg/kg versus 1 mg/kg (of the Ab) (***p < 0.001). (n = 8). (B) PBS versus conjugate at both doses (**p < 0.01). Conjugate 0.2 mg/kg versus 1 mg/kg (of the Ab) (**p < 0.01). (n = 6)
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