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. 2019 Oct 1;30(4):689-705.e6.
doi: 10.1016/j.cmet.2019.07.002. Epub 2019 Jul 25.

Targeting Pathogenic Lafora Bodies in Lafora Disease Using an Antibody-Enzyme Fusion

M Kathryn Brewer  1 , Annette Uittenbogaard  1 , Grant L Austin  1 , Dyann M Segvich  2 , Anna DePaoli-Roach  3 , Peter J Roach  3 , John J McCarthy  4 , Zoe R Simmons  1 , Jason A Brandon  4 , Zhengqiu Zhou  1 , Jill Zeller  5 , Lyndsay E A Young  1 , Ramon C Sun  1 , James R Pauly  6 , Nadine M Aziz  7 , Bradley L Hodges  7 , Tracy R McKnight  7 , Dustin D Armstrong  7 , Matthew S Gentry  8
Affiliations

Targeting Pathogenic Lafora Bodies in Lafora Disease Using an Antibody-Enzyme Fusion

M Kathryn Brewer et al. Cell Metab. .

Abstract

Lafora disease (LD) is a fatal childhood epilepsy caused by recessive mutations in either the EPM2A or EPM2B gene. A hallmark of LD is the intracellular accumulation of insoluble polysaccharide deposits known as Lafora bodies (LBs) in the brain and other tissues. In LD mouse models, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Therefore, LBs have become a therapeutic target for ameliorating LD. Herein, we demonstrate that human pancreatic α-amylase degrades LBs. We fused this amylase to a cell-penetrating antibody fragment, and this antibody-enzyme fusion (VAL-0417) degrades LBs in vitro and dramatically reduces LB loads in vivo in Epm2a-/- mice. Using metabolomics and multivariate analysis, we demonstrate that VAL-0417 treatment of Epm2a-/- mice reverses the metabolic phenotype to a wild-type profile. VAL-0417 is a promising drug for the treatment of LD and a putative precision therapy platform for intractable epilepsy.

Keywords: Lafora bodies; Lafora disease; amylase; antibody-based drug; antibody-enzyme fusion; enzyme therapy; epilepsy; glycogen; glycogen storage disease; metabolomics.

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Conflict of interest statement

Competing interests: D.A. is Chief Scientific Officer and has an equity interest in Valerion Therapeutics. B.L.H. and T.M. are consultants of Valerion Therapeutics. M.S.G. received a sponsored project award from Valerion Therapeutics in accordance with University of Kentucky policies. The other authors declare that they have no competing interests. Valerion Therapeutics has filed one or more patent applications, including WO2018049237A1 of which D.A. is an inventor related to AEF and its use.

Figures

Fig. 1.
Fig. 1.
The antibody-enzyme fusion VAL-0417 degrades starch, a proxy for LBs. (A) Degradation of starch by a panel of amylases. 1 mg of starch was incubated with 5 µg of amylase overnight with constant agitation, then soluble and insoluble fractions were separated by centrifugation. Degradation product in the soluble fractions was quantified by measuring glucose equivalents (i.e. glucan). Reactions were performed in triplicate and data shown are means ± SD. (B) Schematic representation of VAL-0417. The gene encoding the human IgG1 Fab heavy chain fragment (H) was fused to AMY2A, encoding human pancreatic α-amylase, and coexpressed with the gene encoding the human light chain (L) in HEK293–6E cells. Heavy and light chain signal peptides were included to facilitate proper folding and assembly of the Fab fragment. The predicted molecular weight of each polypeptide is shown. (C) Purity of VAL-0417 assessed by reducing (+BME) and nonreducing (-BME) SDS-PAGE. (D) Chemical structure of the α-amylase-specific substrate E-G7-pNP. Reducing (inner) and non-reducing (outer) ends of the substrate are displayed. (E) Degradation of 1 mg starch after a 2-hour incubation with increasing amounts of VAL-0417, expressed as glucan released in the soluble fraction. Mean ±SD of triplicate reactions are shown. (F) Scanning electron micrographs of starch granules in the absence of treatment and after overnight treatment with VAL-0417. Samples were visualized at 2kV by an FE Quanta 250 scanning electron microscope. See also Figure S1.
Fig. 2.
Fig. 2.
A novel protocol for isolating native LBs from LD mice. (A) LB purification scheme. (B) Polysaccharide was purified at different steps in the protocol via the Pflüger method and quantitated via glucose measurement following hydrolysis. Initial tissue weights and total polysaccharide at each step are shown. (C) Polysaccharide yields normalized to tissue weight. Triplicate samples were removed from each fraction and each measured in triplicate. Mean ±SD are shown. (D) Phosphate content of LBs from skeletal muscle and normal rabbit muscle glycogen. Mean ±SD of triplicate measurements are shown. (E) Normalized iodine spectra of purified LBs compared to commercial liver glycogen and amylopectin. Spectra shown are an average of 3 replicates. (F) Brain, (G) heart, and (H) skeletal muscle LBs stained with Lugol’s solution and visualized using a Zeiss Axioimager Z1. See also Figure S2.
Fig. 3.
Fig. 3.
Scanning electron micrographs of isolated LBs. Scanning electron micrographs of LBs purified from brain (A), heart (B), and skeletal muscle (C) displayed using the same scale. LBs from skeletal muscle are also shown at a higher magnification (D). Samples were visualized at 2 kV using an FE Quanta 250. See also Figure S2.
Fig. 4.
Fig. 4.
VAL-0417 degrades LBs in vitro. (A) Degradation of 50 µg LBs from different tissues after overnight incubation with 0, 1, or 10 µg VAL-0417. (B) LBs from different tissues after overnight incubation +/− 10 µg VAL-0417. Insoluble fractions were resuspended post-degradation, stained with Lugol’s solution, and visualized using a Nikon Eclipse E600. (C) Iodine absorbance in pellet fractions after incubation of WT and Epm2a−/− skeletal muscle homogenates +/− 25 µg VAL-0417. In (A), (C), and (E) mean ± SD of triplicates are shown. (D) HPAEC-PAD chromatogram of degradation product after incubation with VAL-0417 for 168 hours. Peak identities were determined based on the elution profile of degradation buffer and glucan standards. The chromatogram is representative of triplicate experiments, and glucose/maltose quantification from the replicates is shown in (E). (E) Quantification of glucose and maltose at various time points throughout the degradation reaction of LBs with VAL-0417. Product released are expressed as a percentage of total LBs. See also Figures S3 and S4.
Fig. 5.
Fig. 5.
VAL-0417 uptake and polysaccharide reduction in cell culture. (A) Rat1 cells and HEK293 cells stably expressing PTG and PP1Cα after 20-hour treatment with increasing concentrations of VAL-0417. VAL-0417 levels were assessed by Western blotting using an anti-AMY2A antibody. ENT2 levels were also quantified by Western blotting. (B) Polysaccharide levels per well in Rat1 and HEK293-PTG/PP1Cα cells after 20-hour treatment with VAL-0417. Data shown are a mean of 4 measurements ± SE. Statistical significance determined by t-test is indicated: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. See also Figure S4F.
Fig. 6.
Fig. 6.
VAL-0417 reduces LB load in vivo after intramuscular (IM) or intravenous (IV) injection. (A) Biodistribution of VAL-0417 levels determined by sandwich ELISA after IM injection of WT mice with PBS or 0.6 mg VAL-0417. (B) Quantification of polysaccharide in the injected gastrocnemii of WT and Epm2a−/− mice after 3 IM injections of PBS or VAL-0417 administered over the course of one week. (C) Quantification of polysaccharide in the heart of WT and Epm2a−/− mice after 4 IV injections of PBS or VAL-0417 administered over a two-week period. Polysaccharides in (B) and (C) were isolated via the Pflüger method and quantified by glucose measurement assays following hydrolysis. Statistical significance is indicated as determined by ANOVA: ** p ≤ 0.01. (D, E) PAS-stained heart tissue of WT (D) and Epm2a−/− (E) mice after IV treatment regimen. Intensely staining PAS-positive deposits are LBs (purple). Tissues were counterstained with hemotoxylin (blue).
Fig. 7.
Fig. 7.
VAL-0417 reduces LB load and reverses metabolic derangement in vivo after continuous ICV infusion. (A) Representative photograph of six 2.0 mm coronal sections. Evans blue dye was injected through the cannula into the lateral ventricle to verify its location within slice 2. (B) Polysaccharide content normalized to protein content in Epm2a−/− brain slices after 28 days of continuous ICV infusion of VAL-0417 or PBS. (C) Total brain polysaccharide normalized to tissue weight from untreated, PBS treated, and VAL-0417 treated Epm2a−/− mice after 8-day ICV infusion. (D) VAL-0417 levels in PBS and VAL-0417 treated animals determined by ELISA. In (B), (C), and (D) at least three technical replicates were performed for each isolated slice to determine an average per slice per animal, and data shown are the mean from each treatment group ± SE. The numbers of animals in each treatment group (n) are shown. Statistical significance is indicated as determined by ANOVA (B) and t-test (C): * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (E) PAS-stained brain slices of PBS and VAL-0417 treated Epm2a−/− mice after 8-day ICV infusion. LB deposits appear purple; tissue was counterstained with hemotoxylin (blue). (F) Two-dimensional principle component (PC) plots for polar metabolites of PBS and VAL-0417 treated Epma2−/− animals (orange and blue dots, respectively) after 8-day infusion compared to untreated WT and Epm2a−/− controls (black and red dots, respectively). There is a clear separation between the groups, which has been shaded accordingly. (G) Relative abundance of mono-, di- and tri-saccharides determined by GCMS from untreated WT and Epm2a−/− mice and also Epm2a−/− mice treated with PBS or VAL-0417. Statistical significance was determined by two-way ANOVA: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. See also Figures S5, S6 and S7.
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