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. 2017 May 31;8:15314.
doi: 10.1038/ncomms15314.

Engineered Clearing Agents for the Selective Depletion of Antigen-Specific Antibodies

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Free PMC article

Engineered Clearing Agents for the Selective Depletion of Antigen-Specific Antibodies

Siva Charan Devanaboyina et al. Nat Commun. .
Free PMC article

Abstract

Here we have designed a novel class of engineered antibody-based reagents ('Seldegs') that induce the selective degradation of antigen-specific antibodies. We demonstrate the rapid and specific clearance of antibodies recognizing the autoantigen, myelin oligodendrocyte glycoprotein and tumour target, HER2. Seldegs have considerable potential in multiple areas, including the treatment of antibody-mediated autoimmunity and diagnostic imaging.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. TZB and 8-18C5 are rapidly cleared by Seldegs in transgenic mice expressing huFcγRs.
(a) Schematic representation of Seldeg design. (b) A concentration of 100 nM MOG-Seldeg or MOG-WT was injected over immobilized mouse FcRn at the indicated pH. (c) Mice were intravenously injected with radiolabelled (125I) 8-18C5 (15 μg) and 24 h later PBS, MOG-WT (31 μg) or MOG-Seldeg (4-fold (31 μg) or 16-fold (125 μg) molar excess; low or high dose, respectively) was delivered intravenously. Radioactivity levels were determined at the indicated times. Whole body or blood levels obtained immediately before Seldeg or control delivery were taken as 100%. (d,e) The same methodology as in c was used, except that radiolabelled TZB (15 μg) injection was followed 24 h later by intravenous delivery of PBS, HER2-WT (51 μg), HER2-Seldeg (51 μg), MOG-Seldeg (31 μg) or Abdeg (MST-HN; 60 μg), each at fourfold molar excess. Error bars indicate s.d. and statistically significant differences are indicated for MOG-WT versus MOG-Seldeg (low) (c) HER2-Seldeg versus MOG-Seldeg (d) and HER2-Seldeg versus Abdeg (e) by *(P<0.05, two-way analysis of variance with Tukey's multiple comparisons; n=6 mice per group). Data shown are representative of two independent experiments.
Figure 2
Figure 2. Seldegs increase the accumulation of antigen-specific antibodies in human endothelial (HMEC-1) cells expressing FcRn-GFP.
(a) HMEC-1 cells were incubated with 100 nM Alexa 647-labelled 8-18C5 (MOG-specific) or TZB (HER2-specific) in complex with 400 nM MOG-Seldeg/MOG-WT or HER2-Seldeg/HER2-WT for 30 min and chased for 0 (30' P) or 60 min (30' P, 60' C). Mean fluorescence intensities (MFI) of Alexa 647-labelled 8-18C5 or TZB for triplicate samples were determined by flow cytometry. Error bars indicate s.d. (b,c) HMEC-1 cells were plated on coverslips and treated as in a, except that Seldegs or control WT proteins were labelled with Alexa 555 and cells were fixed for microscopy. Images of representative cells from multiple cells analysed are shown with GFP, Alexa 555 and Alexa 647 in overlays pseudocoloured green, red and blue, respectively. Representative endosomes in the insets are cropped and expanded. (d) HMEC-1 cells were pre-pulsed with Alexa 555-labelled dextran for 2 h, washed and pulsed with 8-18C5 in complex with MOG-Seldeg, MOG-WT and HER2-Seldeg (concentrations and labels as for a) for 30 min, followed by an 8 h chase. Cells were washed, fixed and imaged, and images for a representative cell from multiple cells analysed are presented. Representative lysosomes in the insets are cropped and expanded. For the overlay, GFP, Alexa 555 and Alexa 647 are pseudocoloured as in b. For bd, scale bars=5 μm, and for insets, scale bars=0.25 μm. Data shown are representative of at least two independent experiments.

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