Immune Responses and Hypercoagulation in ERT for Pompe Disease Are Mutation and rhGAA Dose Dependent

<div><p>Enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) is the only FDA approved therapy for Pompe disease. Without ERT, severely affected individuals (early onset) succumb to the disease within 2 years of life. A spectrum of disease severity and progression exists depending upon the type of mutation in the GAA gene (<i>GAA</i>), which in turn determines the amount of defective protein produced and its enzymatic activity. A large percent of the early onset patients are also cross reactive immunological material negative (CRIM-) and develop high titer immune responses to ERT with rhGAA. New insights from our studies in pre-clinical murine models reveal that the type of <i>Gaa</i> mutation has a profound effect on the immune responses mounted against ERT and the associated toxicities, including activation of clotting factors and disseminated intravascular coagulation (DIC). Additionally, the mouse strain affects outcomes, suggesting the influence of additional genetic components or modifiers. High doses of rhGAA (20 mg/kg) are currently required to achieve therapeutic benefit. Our studies indicate that lower enzyme doses reduce the antibody responses to rhGAA, reduce the incidence of immune toxicity and avoid ERT-associated anaphylaxis. Therefore, development of rhGAA with increased efficacy is warranted to limit immunotoxicities.</p></div

Vital signs measured by pulse oxymetry, prior to and 5 minutes after the 8^th rhGAA IV injection in wt BALB/c and wt 129SVJ mice (n = 5).

<p><b>A</b>) Percentage oxygen saturation <b>B</b>) Heart rate <b>C</b>) Breath distention <b>D</b>) Pulse distention, p<0.05 *, ns = not significant, (inj; injection).</p

Antibody responses to varying doses of rhGAA in null mutation (n = 6) or P545L mutant mice (n = 5).

<p><b>A</b>) Anti-rhGAA IgG1 in 1 mg/kg rhGAA injected GAA-/- 129SVE mice tested weekly <b>B</b>) Anti-rhGAA IgG1 in 5 mg/kg rhGAA injected GAA-/- 129SVE mice <b>C</b>) Anti-rhGAA IgG1 in 20 mg/kg rhGAA injected GAA-/- 129SVE mice <b>D</b>) Anti-rhGAA IgG1 response in 20 mg/kg rhGAA injected P545L mice <b>E</b>) Anti-rhGAA IgG2a in 1 mg/kg rhGAA injected GAA-/- 129SVE mice <b>F</b>) Anti-rhGAA IgG2a in 5 mg/kg rhGAA injected GAA-/- 129SVE mice <b>G</b>) Anti-rhGAA IgG2a in 20 mg/kg rhGAA injected GAA-/- 129SVE mice <b>H</b>) Anti-rhGAA IgG2a in 20 mg/kg rhGAA injected P545L mice. Arrows indicate fold decrease over corresponding 20 mg/kg cohort time point. p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Immune Responses and Hypercoagulation in ERT for Pompe Disease Are Mutation and rhGAA Dose Dependent - Figure 6

<p><b>A</b>) Reduction of clotting times measured in activated partial thromboplastin time (aPTT) in GAA-/- 129SVE and P545L mice. <b>B</b>) D-Dimer levels in GAA-/-129SVE naïve and rhGAA injected mice. Changes in hemorheologic values from complete blood counts in 129SVE GAA-/- mice and P545L naive and 20 mg/kg rhGAA injected mice (5 min post rhGAA injection). <b>C</b>) Platelet count, <b>D</b>) Mean platelet volume, <b>E</b>) Platelet width distribution, p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Comparison of pulse oxymetry measurements of vital signs prior to and post the 4^th rhGAA (1 mg/kg, 5 mg/kg or 20 mg/kg) ERT IV injection in GAA-/- 129SVE (n = 6) and P545L mice (20 mg/kg rhGAA; n = 5).

<p>A) oxygen saturation B) heart rate C) pulse distention D) breath distention, E) Time taken for the formation of a platelet plug post-injury (tail-snip) prior to and post-rhGAA IV administration. Changes in hemorheologic values from complete blood counts in 129SVE GAA-/- mice and P545L naive and 20 mg/kg rhGAA injected mice (5 min post rhGAA injection). F) Hematocrit, G) Hemoglobin, p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Temperature measurements prior to and post rhGAA IV injections in A-C) GAA-/-129SVE mice (n = 6) receiving 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA IV D) GAA-/-129SVE mice injected with PBS E) P545L mice (n = 5) receiving 20 mg/kg of rhGAA IV, F) Experimental timeline indicating GAA-/- 129SVE mice or P545L C57BL/6 x 129SVE mice injected with 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA G) Survival curve.

<p>Temperature measurements prior to and post rhGAA IV injections in A-C) GAA-/-129SVE mice (n = 6) receiving 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA IV D) GAA-/-129SVE mice injected with PBS E) P545L mice (n = 5) receiving 20 mg/kg of rhGAA IV, F) Experimental timeline indicating GAA-/- 129SVE mice or P545L C57BL/6 x 129SVE mice injected with 1 mg/kg, 5 mg/kg or 20 mg/kg doses of rhGAA G) Survival curve.</p

Representative examples of hematoxylin and eosin (H&E) staining of paraffin embedded sections of liver, kidney and heart (n = 3) in A-C) naïve GAA-/- 129SVE mice and D-F) 20 mg/kg rhGAA IV injected GAA-/- 129SVE indicating residual RBC, G-I) 20 mg/kg rhGAA IV injected P545L mice.

<p>Representative examples of hematoxylin and eosin (H&E) staining of paraffin embedded sections of liver, kidney and heart (n = 3) in A-C) naïve GAA-/- 129SVE mice and D-F) 20 mg/kg rhGAA IV injected GAA-/- 129SVE indicating residual RBC, G-I) 20 mg/kg rhGAA IV injected P545L mice.</p

Co-administration of AT2220 promotes greater tissue uptake of rhGAA in GAA KO mice.

<p>Twelve-week old male <i>GAA</i> KO mice were administered vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 oral administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 7 days after the last (<i>i.e.</i>, 4^th) rhGAA administration and tissue GAA activity was measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. Each bar represents the mean±SEM of the GAA activity measured from 5 mice per group. Statistically significant increases were seen in GAA activity compared to baseline (*p<0.05, t-test) and compared to rhGAA administration alone (#p<0.05, t-test). For comparison, GAA levels in wild-type C57BL/6 mice were 15±2, 16±0.6, 21±3, 18±2, 11±2, and 25±3 nmol/mg protein/hr in heart, diaphragm, gastrocnemius, quadriceps, triceps, and tongue, respectively (mean±SEM of 7 mice).</p

Co-administration of AT2220 promotes cell type-specific reduction of glycogen in GAA KO mice.

<p>Twelve-week old male <i>GAA</i> KO mice were administered vehicle (water) or AT2220 (30 mg/kg) via oral gavage once every other week for 8 weeks. Thirty minutes after each AT2220 oral administration, vehicle (saline) or rhGAA (20 mg/kg) was administered via bolus tail vein injection. Mice were euthanized 21 days after the last (<i>i.e.,</i> 4^th) rhGAA administration and glycogen levels in heart and quadriceps were measured immunohistochemically as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040776#s2" target="_blank">Materials and Methods</a>’. A strong glycogen signal, represented as dark blue or pink spots (denoted with arrows) in heart and quadriceps, respectively, was observed. (*) indicates glycogen reduction in individual skeletal muscle fibers of quadriceps. The data shown are representative photomicrographs from 6 mice/group (magnification: 20X).</p

AT2220 increases the physical stability of rhGAA in vitro.

<p>(<b>A</b>) Time course of rhGAA denaturation in neutral and acidic buffer at 37°C in the absence and presence of 50 µM AT2220. Denaturation was monitored by changes in the fluorescence of SYPRO Orange as a function of time. (<b>B</b>) Time course of rhGAA inactivation (<i>i.e.</i> loss of activity) in neutral and acidic buffer at 37°C in the absence and presence of 50 µM AT2220. (<b>C</b>) Time course of rhGAA inactivation (<i>i.e.</i> loss of activity) in human whole blood at 37°C in the absence and presence of 50 µM AT2220. In both (<b>B</b>) and (<b>C</b>), GAA enzyme activity was determined at the indicated time points using the fluorogenic substrate 4-MUG. To obtain relative enzyme activity levels, measurements at the various time points were compared to the activity at the zero time point.</p