- Email: [email protected]
2 study
Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
Recombinant α-galactosidase A (rh-αGAL-A) is used in enzyme replacement therapy (ERT) to treat Fabry disease (FD). Though ERT is shown to have high efficacy, there is a significant mortality rate due to cardiac, renal, and cerebrovascular end-organ injury. This suggests that cell/organ-type specific uptake of rh-αGAL-A can be key for delivery recombinant enzyme. We investigated the uptake of rh-αGAL-A in vitro in cell types affected in Fabry disease, both from healthy patients and those with FD: primary: fibroblasts, podocytes and peripheral blood mononuclear cells (PBMC) and cell lines: HEK293, THP-1, HUVEK. Enzyme uptake was measured by α-galactosidase assay. Because CI-M6PR is the main receptor involved in the enzyme delivery, inhibitors of caveolae (Filipin and Genistin) and clathrin (chloroquine), microtubule depolymerization (nocodazole) were used to study the endocytosis. To explore mechanisms of uptake, western blot analysis was done using α-Gal A, LAMP1 (lysosomal-associated membrane protein 1), as well as CI-M6PR (cation-independent mannose 6 phosphate receptor) antibodies. Data shows that the efficiency of enzyme uptake and maximum capacity is time and cell type specific. The maximum uptake capacity for the rhaGAL-A in fibroblasts was exceeded by 70-fold compared to 15-fold for podocytes and 6-fold for HEK293. We observed lower concentrations of endogenous enzyme indicating more efficient cellular uptake. Moreover, the enzyme activity level didnot correlated with level of uptake rh-aGAL-A protein. Transport of rh-αGAL-A into lysosomes during the initial hours is achieved by clathrin and caveolae-mediated endocytosis, as evidenced by the inhibition in presence of Filipin, Genistin, and chloroquine. Microtubule depolymerazation partially blocked the uptake of rh-aGAL-A. Conclusions: efficiency of enzyme uptake, but not maximum enzyme activity, is time and cell type specific. The cation independent CI-M6PR plays an important role in the delivery of rh-α Gal A to the lysosome via clatrinin and caveolae-mediated endocytosis.
doi:10.1016/j.ymgme.2016.11.062
54 A randomized open-label clinical trial of intrathecal recombinant human alpha-L-iduronidase for cognitive decline in mucopolysaccharidosis type I Agnes Chena, Igor Nestrasilb, Elsa Shapiroc, Julie Eisengartb, Timothy Maarupa, Lynda Polgreena, Paul Harmatzd, Patricia Dicksona, aLos Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, United States, bUniversity of Minnesota, Minneapolis, MN, United States, cShapiro and Delaney, LLC, Minneapolis, MN, United States, dUCSF Benioff Children's Hospital Oakland Research Institute, Oakland, CA, United States Mucopolysaccharidosis (MPS) I causes progressive loss of intellect, memory, and attention even in the attenuated forms of the disease. We conducted a randomized, open-label study of recombinant human alpha-L-iduronidase administered intrathecally to 8 subjects with MPS I over the period of time from 2009-2015. Eligible subjects were age 6 years or older with neuropsychological impairment defined as a score that was at least one standard deviation below the mean in at least one domain on standardized testing. Subjects were excluded if they had undergone hematopoietic stem cell transplantation with engraftment, were pregnant or considering pregnancy, had contraindications to lumbar spinal tap, had severely impaired CSF flow, or were unable to comply with study procedures, including the ability to perform cognitive testing. The study was designed at 50:50 randomization to treatment or no treatment for a period of one year. Treated patients received 1.74 mg recombinant human alpha-L-iduronidase diluted in 6 mL Elliott’s B
S35
artificial cerebrospinal fluid solution (total volume 9 mL) administered via lumbar spinal injection. After one year, all subjects were enrolled in treatment. The schedule involved four monthly doses followed by dosing at three month intervals for the duration of the subject’s participation in the study. We collected cerebrospinal fluid for routine chemistries, glycosaminoglycans, and anti-iduronidase antibody. Outcome measures included neurological exam, neuropsychological testing, and advanced brain MRI imaging. Subjects experienced 14 serious adverse events, one possibly related to study drug or procedures, which was a headache that required one extra day of hospitalization. There were 75 nonserious adverse events. Adverse events possibly related to IT ERT included low back pain, groin pain, neck stiffness, headache, transient blurry vision, and buttock pain. This study describes the investigational use of intrathecal recombinant human alpha-L-iduronidase, a potential therapy for central nervous system disease due to MPS I.
doi:10.1016/j.ymgme.2016.11.063
55 Immunogenicity to cerliponase alfa, an enzyme replacement therapy for patients with CLN2 disease: results from a phase 1/2 study Anu Cherukuri, Heather Cahan, Andrea Van Tuyl, Greg de Hart, Peter Slasor, Laurie Bray, Josh Henshaw, Temitayo Ajayi, Dave Jacoby, Charles O'Neill, Becky Schweighardt, BioMarin Pharmaceutical Inc., Novato, CA, United States The immunogenic potential of an enzyme replacement therapeutic (ERT) delivered via an intracerebroventricular (ICV) route for treatment of a neurological disease has not been previously reported. Recombinant human tripeptidyl peptidase-1 (TPP1), cerliponase alfa, is in development by BioMarin Pharmaceutical as a potential therapy for CLN2, a rapidly progressing neurodegenerative disease characterized by the deficiency of TPP1, typically leading to death in the first or second decade of life. Results from a Phase 1/2 clinical study demonstrate that cerliponase alfa induces anti-drug antibody (ADA) production in the serum and cerebrospinal fluid (CSF) of 19/24 (79%) and 5/24 (21%) of the study participants, respectively. The magnitude of the ADA response varied in these two compartments with the peak response in the CSF being ~100-fold lower in magnitude than that detected in the serum, despite exposure to the drug being significantly higher in the CSF. Serum ADA titer was sustained in 12/ 19 (63%), declined in 5/19 (26%) and reverted to undetectable levels in 2/19 (11%) subjects by Week 109. CSF ADA titer was sustained in 2/5 (40%) while it declined to undetectable levels in 3/5 (60%) subjects by Week 109. Further, neutralizing antibodies were not detected in the CSF of any of the subjects through Week 109. Notably, we report that neither serum ADA titer nor drug-specific IgE outcome were associated with incidence or severity of hypersensitivity adverse events after ICV administered ERT treatment and no events of anaphylaxis were reported. No association was found between serum or CSF ADA titers and the primary efficacy measure, change from baseline CLN2 score. Taken together, the data indicate that the immunogenicity profile elicited by ICV treatment with cerliponase alfa for N100 weeks was characterized by sustained CSF ADA titers in b10% of subjects and was not associated with adverse impacts on safety or treatment outcome.
doi:10.1016/j.ymgme.2016.11.064
Recombinant α-galactosidase A (rh-αGAL-A) is used in enzyme replacement therapy (ERT) to treat Fabry disease (FD). Though ERT is shown to have high efficacy, there is a significant mortality rate due to cardiac, renal, and cerebrovascular end-organ injury. This suggests that cell/organ-type specific uptake of rh-αGAL-A can be key for delivery recombinant enzyme. We investigated the uptake of rh-αGAL-A in vitro in cell types affected in Fabry disease, both from healthy patients and those with FD: primary: fibroblasts, podocytes and peripheral blood mononuclear cells (PBMC) and cell lines: HEK293, THP-1, HUVEK. Enzyme uptake was measured by α-galactosidase assay. Because CI-M6PR is the main receptor involved in the enzyme delivery, inhibitors of caveolae (Filipin and Genistin) and clathrin (chloroquine), microtubule depolymerization (nocodazole) were used to study the endocytosis. To explore mechanisms of uptake, western blot analysis was done using α-Gal A, LAMP1 (lysosomal-associated membrane protein 1), as well as CI-M6PR (cation-independent mannose 6 phosphate receptor) antibodies. Data shows that the efficiency of enzyme uptake and maximum capacity is time and cell type specific. The maximum uptake capacity for the rhaGAL-A in fibroblasts was exceeded by 70-fold compared to 15-fold for podocytes and 6-fold for HEK293. We observed lower concentrations of endogenous enzyme indicating more efficient cellular uptake. Moreover, the enzyme activity level didnot correlated with level of uptake rh-aGAL-A protein. Transport of rh-αGAL-A into lysosomes during the initial hours is achieved by clathrin and caveolae-mediated endocytosis, as evidenced by the inhibition in presence of Filipin, Genistin, and chloroquine. Microtubule depolymerazation partially blocked the uptake of rh-aGAL-A. Conclusions: efficiency of enzyme uptake, but not maximum enzyme activity, is time and cell type specific. The cation independent CI-M6PR plays an important role in the delivery of rh-α Gal A to the lysosome via clatrinin and caveolae-mediated endocytosis.
doi:10.1016/j.ymgme.2016.11.062
54 A randomized open-label clinical trial of intrathecal recombinant human alpha-L-iduronidase for cognitive decline in mucopolysaccharidosis type I Agnes Chena, Igor Nestrasilb, Elsa Shapiroc, Julie Eisengartb, Timothy Maarupa, Lynda Polgreena, Paul Harmatzd, Patricia Dicksona, aLos Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, United States, bUniversity of Minnesota, Minneapolis, MN, United States, cShapiro and Delaney, LLC, Minneapolis, MN, United States, dUCSF Benioff Children's Hospital Oakland Research Institute, Oakland, CA, United States Mucopolysaccharidosis (MPS) I causes progressive loss of intellect, memory, and attention even in the attenuated forms of the disease. We conducted a randomized, open-label study of recombinant human alpha-L-iduronidase administered intrathecally to 8 subjects with MPS I over the period of time from 2009-2015. Eligible subjects were age 6 years or older with neuropsychological impairment defined as a score that was at least one standard deviation below the mean in at least one domain on standardized testing. Subjects were excluded if they had undergone hematopoietic stem cell transplantation with engraftment, were pregnant or considering pregnancy, had contraindications to lumbar spinal tap, had severely impaired CSF flow, or were unable to comply with study procedures, including the ability to perform cognitive testing. The study was designed at 50:50 randomization to treatment or no treatment for a period of one year. Treated patients received 1.74 mg recombinant human alpha-L-iduronidase diluted in 6 mL Elliott’s B
S35
artificial cerebrospinal fluid solution (total volume 9 mL) administered via lumbar spinal injection. After one year, all subjects were enrolled in treatment. The schedule involved four monthly doses followed by dosing at three month intervals for the duration of the subject’s participation in the study. We collected cerebrospinal fluid for routine chemistries, glycosaminoglycans, and anti-iduronidase antibody. Outcome measures included neurological exam, neuropsychological testing, and advanced brain MRI imaging. Subjects experienced 14 serious adverse events, one possibly related to study drug or procedures, which was a headache that required one extra day of hospitalization. There were 75 nonserious adverse events. Adverse events possibly related to IT ERT included low back pain, groin pain, neck stiffness, headache, transient blurry vision, and buttock pain. This study describes the investigational use of intrathecal recombinant human alpha-L-iduronidase, a potential therapy for central nervous system disease due to MPS I.
doi:10.1016/j.ymgme.2016.11.063
55 Immunogenicity to cerliponase alfa, an enzyme replacement therapy for patients with CLN2 disease: results from a phase 1/2 study Anu Cherukuri, Heather Cahan, Andrea Van Tuyl, Greg de Hart, Peter Slasor, Laurie Bray, Josh Henshaw, Temitayo Ajayi, Dave Jacoby, Charles O'Neill, Becky Schweighardt, BioMarin Pharmaceutical Inc., Novato, CA, United States The immunogenic potential of an enzyme replacement therapeutic (ERT) delivered via an intracerebroventricular (ICV) route for treatment of a neurological disease has not been previously reported. Recombinant human tripeptidyl peptidase-1 (TPP1), cerliponase alfa, is in development by BioMarin Pharmaceutical as a potential therapy for CLN2, a rapidly progressing neurodegenerative disease characterized by the deficiency of TPP1, typically leading to death in the first or second decade of life. Results from a Phase 1/2 clinical study demonstrate that cerliponase alfa induces anti-drug antibody (ADA) production in the serum and cerebrospinal fluid (CSF) of 19/24 (79%) and 5/24 (21%) of the study participants, respectively. The magnitude of the ADA response varied in these two compartments with the peak response in the CSF being ~100-fold lower in magnitude than that detected in the serum, despite exposure to the drug being significantly higher in the CSF. Serum ADA titer was sustained in 12/ 19 (63%), declined in 5/19 (26%) and reverted to undetectable levels in 2/19 (11%) subjects by Week 109. CSF ADA titer was sustained in 2/5 (40%) while it declined to undetectable levels in 3/5 (60%) subjects by Week 109. Further, neutralizing antibodies were not detected in the CSF of any of the subjects through Week 109. Notably, we report that neither serum ADA titer nor drug-specific IgE outcome were associated with incidence or severity of hypersensitivity adverse events after ICV administered ERT treatment and no events of anaphylaxis were reported. No association was found between serum or CSF ADA titers and the primary efficacy measure, change from baseline CLN2 score. Taken together, the data indicate that the immunogenicity profile elicited by ICV treatment with cerliponase alfa for N100 weeks was characterized by sustained CSF ADA titers in b10% of subjects and was not associated with adverse impacts on safety or treatment outcome.
doi:10.1016/j.ymgme.2016.11.064