Research ArticleALS

A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis

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Science Translational Medicine  05 Dec 2018:
Vol. 10, Issue 470, eaah3924
DOI: 10.1126/scitranslmed.aah3924
  • Fig. 1 Human α-miSOD1 antibody targets an epitope in pathologically misfolded SOD1.

    Human α-miSOD1 antibody targets an epitope in pathologically misfolded SOD1 that is exposed in motor neurons of patients with FALS and SALS. (A) In a direct enzyme-linked immunosorbent assay (ELISA), α-miSOD1 bound with high affinity to denatured (EC50, ~70 pM) and oxidized misfolded (EC50, ~600 pM) human SOD1, while showing only minimal binding to the physiological SOD1 dimers. (B) In a direct ELISA, α-miSOD1 bound with similar affinity to the short bovine serum albumin (BSA)–coupled SOD1 76-82 peptide containing the minimal α-miSOD1 epitope (SOD1 73-83 peptide; EC50, ~30 pM; SOD1 76-82 peptide; EC50, ~13 pM) compared to denatured (EC50, ~33 pM) human SOD1. (C) α-miSOD1 detected misfolded SOD1 in the ventral horn of lumbar spinal cord sections in the majority of patients with SALS and patients with FALS carrying mutant SOD1 as well as selected patients with FALS carrying non-SOD1 mutations or carrying C9ORF72 mutations compared to non-neurological controls (NNC; scale bar, 50 μm). (D to G) Quantification of the area positive for α-miSOD1 binding within the cell soma of ventral horn motor neurons (intraneuronal) is shown. (D) Shown is staining of spinal cord sections from patients with SALS (n = 98), FALS non-SOD1 patients (n = 20), and FALS SOD1 patients (n = 3) relative to the staining of spinal cord sections from NNC patients (n = 41). A reduction in neuron number (E) and neuron size (F) was observed in the SALS and FALS non-SOD1 mutant group. The total parenchymal-positive area for α-miSOD1 binding (G) was increased in all groups of patients with ALS. Kruskal-Wallis, Dunn’s multiple comparison test, **P < 0.01, ****P < 0.0001. OD450 nm, optical density at 450 nm.

  • Fig. 2 α-miSOD1 selectively targets pathologically misfolded SOD1 in the spinal cords of SODG93A and SODG37R transgenic mice.

    (A) Shown is SOD1 pathology in the spinal cords of SODG93A and SOD1G37R transgenic mice in the terminal stages of disease stained with α-miSOD1 antibody. (B) α-miSOD1 immunoreactivity was detectable in the spinal cords of SOD1G93A transgenic mice at 30 days of age. These α-miSOD1–positive dense aggregates increased with age as motor impairments progressed, with additional staining appearing in vacuolized axonal structures (black arrows) at the terminal stage of disease (scale bar, 100 μm). No staining was detectable in the spinal cords from age-matched wild-type mice. (C) Immunoprecipitation (IP) with α-miSOD1 antibody captured SOD1 specifically in spinal cord but not in kidney homogenates of SODG93A and SOD1G37R transgenic mice. A pan-SOD1 antibody captured SOD1 in both tissues. (D) α-miSOD1 immunoprecipitated SOD1 in spinal cord homogenates from 160-day-old SODG93A mice in the terminal stages of disease and, to a lesser extent, in 40-day-old SODG93A animals; no SOD1 was precipitated from kidney homogenates. The pan-SOD1 antibody (Sigma-Aldrich S2147) captured SOD1 in all three homogenates tested, whereas the isotype control antibody did not pull down SOD1. (E) α-miSOD1 detected NP40 detergent–insoluble SOD1 aggregates that accumulated in an age-dependent and tissue-specific manner as revealed by filter retardation assays. WB, Western blot.

  • Fig. 3 Intracerebroventricular treatment of SOD1G93A mice with chα-miSOD1 antibody.

    Intracerebroventricular treatment of SOD1G93A transgenic mice with mouse chimeric chα-miSOD1 antibody increased survival, delayed muscle atrophy, and ameliorated gait symptoms. (A and B) High-speed video gait analysis showed that gait disturbances due to muscle atrophy in the lower body of SOD1G93A transgenic mice were ameliorated by continuous intracerebroventricular infusion of chα-miSOD1 antibody 20 days after the start of treatment. This was reflected by a wider maximum angle between iliac crest and ankle during maximum protraction (A) and a more forward mean horizontal position of the hindlimb toe (B). (C) Treatment of SOD1G93A transgenic mice with chα-miSOD1 antibody from 60 days of age onward increased median survival by 7 days (P < 0.05, log-rank Mantel-Cox test). (D) Delayed muscle atrophy led to a higher body weight in chα-miSOD1-treated SOD1G93A transgenic mice. (E and F) Misfolded SOD1 in the ventral horn of the lumbar spinal cord was reduced at the end stage of disease by treatment with chα-miSOD1 antibody (scale bar, 400 μm; n = 14 for the vehicle-treated group and n = 10 for the chα-miSOD1–treated group). (G to J) Treatment with chα-miSOD1 antibody resulted in a trend toward lower numbers of Iba1-positive microglia (brown, H and I) and an increased percentage of microglia associated with SOD1 aggregates (blue, asterisks, H and J) compared to vehicle-treated control animals. (K) Drug concentrations in blood plasma and spinal cord homogenates are shown 2, 5, 10, and 20 days after 100 mg/kg ip injection of human α-miSOD1 antibody in 3-month-old SOD1G93A transgenic mice (n = 4 for each subgroup). (L to N) Human α-miSOD1 in vivo binding detects structures in the lumbar spinal cord containing high concentrations of misfolded SOD1 such as vacuolized axons and supernumerary axonal structures (black arrows), which were labeled in independent α-miSOD1 (M and N) but not in isotype control antibody–treated (L) SOD1G93A transgenic animals. *P < 0.05, t test.

  • Fig. 4 Weekly intraperiteoneal injections of chα-miSOD1 increase survival of SOD1G37R transgenic mice.

    Treatment with weekly intraperitoneal injections of mouse chimeric chα-miSOD1 antibody from 90 days of age onward increased survival of SOD1G37R transgenic mice and ameliorated motor symptoms, muscle atrophy, and loss of motor neurons. (A) chα-miSOD1 treatment delayed the median onset of motor symptoms on the rotarod test by 49 days compared to vehicle-treated animals or by 55 days compared to isotype control antibody–treated animals (mean disease onset, chα-miSOD1–treated mice, 486 days; vehicle-treated mice, 437 days; isotype antibody–treated mice, 431 days; P < 0.001, log-rank Mantel-Cox test). (B) Median survival of chα-miSOD1–treated mice increased by 59 days compared to vehicle-treated control mice or by 36 days compared to isotype antibody–treated control animals (median life span of 538 days for chα-miSOD1–treated mice, 479 days for vehicle-treated mice, and 502 days for isotype antibody–treated mice; P < 0.001, log-rank Mantel-Cox test). (C and D) Treatment with chα-miSOD1 antibody delayed the onset of motor symptoms on the rotarod test (C) and in the PaGE test (D) compared to isotype antibody–treated control or vehicle-treated control mice. (E) Number of surviving motor neurons in the lumbar spinal cord was increased by more than twofold in chα-miSOD1–treated mice compared to isotype antibody–treated control or vehicle-treated control mice. (F and G) Treatment with chα-miSOD1 antibody increased gastrocnemius muscle (F) and flexor digitorum longus muscle weight (G) at the disease end point in chα-miSOD1–treated mice compared to both control groups. *P < 0.05, **P < 0.01, ***P < 0.001 [analysis of variance (ANOVA), Bonferroni’s multiple comparison test; t test at individual time points for (C) and (D)].

  • Fig. 5 Weekly intraperitoneal dosing of SOD1G93A transgenic mice with chα-miSOD1 antibody delays disease onset.

    Shown is delayed disease onset, extension of survival, and reduced muscle atrophy after weekly intraperitoneal dosing with 3, 10, or 30 mg/kg chα-miSOD1 antibody in SOD1G93A transgenic mice. (A) A delay in disease onset (peak body weight) was observed with all three doses tested (P < 0.05 for 3 and 10 mg/kg, P < 0.01 for 30 mg/kg, log-rank Mantel-Cox test). (B) Extension of survival was observed with all three doses tested (P < 0.01 for 3 mg/kg and P < 0.001 for 10 and 30 mg/kg, log-rank Mantel-Cox test). (C) Attenuation of muscle atrophy in animals with end-stage disease was observed after chα-miSOD1 treatment at all three doses compared to vehicle-treated control mice (Kruskal-Wallis, Dunn’s post hoc, **P < 0.01; *P < 0.05, t test, n = 25 to 55 per group). (D) Antibody concentrations in plasma correlated linearly with dose after weekly intraperitoneal dosing with chα-miSOD1 antibody. (E) No effect on survival was seen in SOD1G93A transgenic mice after treatment with an aglycosylated version of chα-miSOD1 (30 mg/kg) called chα-miSOD1–agly (n = 18 to 25 per group).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/470/eaah3924/DC1

    Materials and Methods

    Fig. S1. α-miSOD1 is selective for misfolded human SOD1.

    Fig. S2. α-miSOD1 immunohistochemical analysis of human spinal cord (Newcastle brain bank).

    Fig. S3. α-miSOD1 immunohistochemical analysis of human spinal cord (AMC brain bank).

    Fig. S4. α-miSOD1 immunohistochemical analysis of human spinal cord (KSSG and Netherlands brain banks).

    Fig. S5. α-miSOD1 immunohistochemical analysis of human spinal cord (UCSD brain bank).

    Fig. S6. α-miSOD1 immunohistochemical analysis of human spinal cord (MGH and Netherlands brain banks).

    Fig. S7. α-miSOD1 immunoreactivity is associated with glial markers, and α-miSOD1 treatment reduces neuroinflammation.

    Fig. S8. α-miSOD1 binding epitope in the native human SOD1 structure.

    Table S1. Average patient demographics.

    Table S2. Subset analysis of misfolded SOD1 histology in patients with SALS versus NNC.

    Data file S1. Raw data (Excel file).

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. α-miSOD1 is selective for misfolded human SOD1.
    • Fig. S2. α-miSOD1 immunohistochemical analysis of human spinal cord (Newcastle brain bank).
    • Fig. S3. α-miSOD1 immunohistochemical analysis of human spinal cord (AMC brain bank).
    • Fig. S4. α-miSOD1 immunohistochemical analysis of human spinal cord (KSSG and Netherlands brain banks).
    • Fig. S5. α-miSOD1 immunohistochemical analysis of human spinal cord (UCSD brain bank).
    • Fig. S6. α-miSOD1 immunohistochemical analysis of human spinal cord (MGH and Netherlands brain banks).
    • Fig. S7. α-miSOD1 immunoreactivity is associated with glial markers, and α-miSOD1 treatment reduces neuroinflammation.
    • Fig. S8. α-miSOD1 binding epitope in the native human SOD1 structure.
    • Table S1. Average patient demographics.
    • Table S2. Subset analysis of misfolded SOD1 histology in patients with SALS versus NNC.

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    Other Supplementary Material for this manuscript includes the following:

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