Successful Elosulfase Alfa Desensitization Protocol in a Patient With Morquio A Syndrome.

Patients with lysosomal storage diseases may require modifications to standard drug desensitization protocols; personalized medicine as well as development of new treatment options are needed.

Oral immunotherapy tolerizes mice to enzyme replacement therapy for Morquio A syndrome.

Immune response to therapeutic enzymes poses a detriment to patient safety and treatment outcome. Enzyme replacement therapy (ERT) is a standard therapeutic option for some types of mucopolysaccharidoses, including Morquio A syndrome caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Current protocols tolerize patients using cytotoxic immunosuppressives, which can cause adverse effects. Here we show development of tolerance in Morquio A mice via oral delivery of peptide or GALNS for 10 days prior to ERT. Our results show that using an immunodominant peptide (I10) or the complete GALNS enzyme to orally induce tolerance to GALNS prior to ERT resulted in several improvements to ERT in mice: (a) decreased splenocyte proliferation after in vitro GALNS stimulation, (b) modulation of the cytokine secretion profile, (c) decrease in GALNS-specific IgG or IgE in plasma, (d) decreased GAG storage in liver, and (e) fewer circulating immune complexes in plasma. This model could be extrapolated to other lysosomal storage disorders in which immune response hinders ERT.

Enzyme Replacement Therapies and Immunogenicity in Lysosomal Storage Diseases: Is There a Pattern?

Lysosomal storage diseases arise because of genetic mutations that result in nonfunctioning or dysfunctional lysosomal enzymes responsible for breaking down molecules such as glycosaminoglycans or glycogen. Many of these storage diseases, such as the mucopolysaccharidosis (MPS) disorders and Pompe disease, can now be treated with infusion therapies to replace the dysfunctional protein with active enzyme. Although these therapies are effective, in at least one condition, infantile-onset Pompe disease, antibodies that develop against the drug significantly reduce its efficacy. However, this influence on efficacy does not appear to manifest across all enzyme replacement therapies. An example is MPS IVA, or Morquio A syndrome, in which the glycosaminoglycans keratan sulfate and chondroitin-6-sulfate accumulate in tissues as a result of N-acetylgalactosamine-6-sulfatase deficiency. The current approved treatment for MPS IVA is elosulfase alfa, a recombinant human enzyme replacement therapy. Although all patients receiving elosulfase alfa treatment develop antidrug antibodies and most develop neutralizing antibodies, clinical data to date show no effect on drug efficacy or safety. Overall, the relevance of antidrug antibodies specific to enzyme replacement therapies for the lysosomal storage diseases remains a mixed picture that will require time and continued clinical follow-up to resolve for each specific condition and treatment.

Immunogenicity of Elosulfase Alfa, an Enzyme Replacement Therapy in Patients With Morquio A Syndrome: Results From MOR-004, a Phase III Trial.

PURPOSE: Morquio A syndrome (mucopolysaccharidosis IVA [MPS IVA]) is a lysosomal storage disorder caused by deficiency of the enzyme N-acetylgalactosamine-6-sulfatase, which is required to degrade the glycosaminoglycan keratan sulfate. Morquio A is associated with extensive morbidity and early mortality. Elosulfase alfa is an enzyme replacement therapy that provides a treatment option for patients with Morquio A. We examined the immunogenicity profile of elosulfase alfa, assessing any correlations between antidrug antibodies and the efficacy and safety outcomes in 176 patients with Morquio A from a 24-week international Phase III trial. METHODS: Patients were randomized to placebo (n = 59) or elosulfase alfa 2.0 mg/kg administered weekly (n = 58) or every other week (n = 59) as an ~4-hour infusion. Blood samples were routinely tested to determine drug-specific total antibody titer and neutralizing antibody (NAb) positivity. Drug-specific immunoglobulin E positivity was tested routinely and in response to severe hypersensitivity adverse events (AEs). Antidrug antibody positivity and titer were compared with efficacy and safety metrics to assess possible correlations. FINDINGS: The 176 patients in the trial were 54% female, with a mean age of 11.9 years. In all patients treated with elosulfase alfa antidrug antibodies developed, and in the majority, antibodies capable of interfering with cation-independent mannose-6-phosphate receptor binding in vitro (NAb) developed. Less than 10% of patients tested positive for drug-specific IgE during the study. Despite the high incidence of anti-elosulfase alfa antibodies, no correlations were detected between higher total antibody titers or NAb positivity and worsened 6-minute walk test results, urine keratin sulfate levels, or hypersensitivity AEs. Drug-specific IgE positivity had no apparent association with the occurrence of anaphylaxis, other hypersensitivity AEs, and/or treatment withdrawal. IMPLICATIONS: Despite the universal development of antidrug antibodies, elosulfase alfa treatment was both safe and well tolerated and immunogenicity was not associated with reduced treatment effect. ClinicalTrials.gov identifier: NCT01275066. (Clin Ther.

N-acetylgalactosamine-6-sulfatase protein detection in MPS IVA patient and unaffected control samples.

BACKGROUND: Mucopolysaccharidosis type IVA (MPS IVA; Morquio syndrome) is a lysosomal storage disorder caused by a deficiency in the activity of the lysosomal hydrolase N-acetylgalactosamine-6-sulfatase (GALNS). MPS IVA patients can present with severe myelopathy, hearing loss, heart valve involvement, short trunk/dwarfism and corneal clouding. Early diagnosis of MPS IVA will allow potential treatments to be implemented before the onset of irreversible pathology. METHODS: We have developed a sensitive immune-quantification assay for the accurate detection of GALNS protein in skin fibroblasts, blood and plasma from unaffected control and MPS IVA patients. RESULTS: MPS IVA patient fibroblast extracts (n=11) had non-detectable (ND)-10 ng/mg of 6-sulfatase protein compared to 3-82 ng/mg for normal controls (n=19). Dried blood-spots from MPS IVA patients (n=4) contained ND-1.3 ng/L of 6-sulfatase protein compared to 18-145 ng/L for normal controls (n=49). Plasma from MPS IVA patients (n=7) contained ND 6-sulfatase protein compared to 1-9 ng/L for normal controls (n=49). CONCLUSIONS: The immune assay described here had the capacity to accurately measure the amount of GALNS protein in various biological samples, providing the basis of an assay that could be further developed to enable newborn and high-risk population screening for MPS IVA patients.

Development of MPS IVA mouse (Galnstm(hC79S.mC76S)slu) tolerant to human N-acetylgalactosamine-6-sulfate sulfatase.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disease caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. In recent studies of enzyme replacement therapy for animal models with lysosomal storage diseases, cellular and humoral immune responses to the injected enzymes have been recognized as major impediments to effective treatment. To study the long-term effectiveness and side effects of therapies in the absence of immune responses, we have developed an MPS IVA mouse model, which has many similarities to human MPS IVA and is tolerant to human GALNS protein. We used a construct containing both a transgene (cDNA) expressing inactive human GALNS in intron 1 and an active site mutation (C76S) in adjacent exon 2 and thereby introduced both the inactive cDNA and the C76S mutation into the murine Galns by targeted mutagenesis. Affected homozygous mice have no detectable GALNS enzyme activity and accumulate glycosaminoglycans in multiple tissues including visceral organs, brain, cornea, bone, ligament and bone marrow. At 3 months, lysosomal storage is marked within hepatocytes, reticuloendothelial Kupffer cells, and cells of the sinusoidal lining of the spleen, neurons and meningeal cells. The bone storage is also obvious, with lysosomal distention in osteoblasts and osteocytes lining the cortical bone, in chondrocytes and in the sinus lining cells in bone marrow. Ubiquitous expression of the inactive human GALNS was also confirmed by western blot using the anti-GALNS monoclonal antibodies newly produced, which resulted in tolerance to immune challenge with human enzyme. The newly generated MPS IVA mouse model should provide a good model to evaluate long-term administration of enzyme replacement.

Mucopolysaccharidosis IVA (Morquio A): identification of novel common mutations in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene in Italian patients.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed by RT-PCR with one amplicon and direct sequence analyses using cDNA samples from 15 Italian MPS IVA patients. Each mutation was confirmed at the genomic level. In this study, 13 different gene mutations with four common mutations (over 10% of mutant alleles) were identified in 12 severe and three milder (attenuated) MPS IVA patients. The gene alterations in 12 out of 13 were found to be point mutations and only one mutation was deletion. Ten of 13 mutations were novel. The c.1070C>T (p.Pro357Leu) mutation coexisted with c.1156C>T (p.Arg386Cys) mutation on the same allele. Together they accounted for 100% of the 30 disease alleles of the patients investigated. Four common mutations accounted for 70% of mutant alleles investigated. Urine keratan sulfate (KS) concentrations were elevated in all patients investigated. These data provide further evidence for extensive allelic heterogeneity and importance of relation among genotype, phenotype, and urine KS excretion as a biomarker in MPS IVA.

Production and characterization of monoclonal antibodies to shark cartilage proteoglycan

1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polydisperse and large (molecular mass: 1-10 x 10**6 Daltons) and contain chondroitin sulfate and keratan sulfate side chains, but PG2 is somewhat smaller tham PG1 and contains less keratan sulfate. 2. Monoclonal antibodies were raised against PG1. Many antibodies were obtained and one of them, MST1, was subcloned and furter characterized. This monoclonal antibody reacts with PG1 and PG2 from shark cartilage and also with aggrecan from bovine trachea cartilage. Chondroitinase AC-treated proteglycans react MST1, indicating that the antibody does not reconize chondroitin sulfate. MST1 also recognizes aggrecan from human cartilage and a proteoglycan from bovine brain (neurocan) but it does reconize proteoglycans from rat Walker tumor, fetal calf muscle and decorin from human myoma. 3. Using MST1 we were able to demonstrate that both PG1 aggregate with hyaluronic acid

Morquio's disease type A: absence of material cross reacting with antibodies against N-acetylgalactosamine-6-sulfate sulfatase.

An antiserum was raised in guinea pigs against purified normal human N-acetylgalactosamine-6-sulfate sulfatase, the enzyme affecting in Morquio's disease type A. The antiserum precipitated most of N-acetylgalactosamine-6-sulfate sulfatase from a concentrate of normal human urine. The antigen-antibody complex was enzymatically active. Urine concentrates from five patients with Morquio's disease type A did not contain material competing with the normal enzyme for binding to soluble or Sepharose-bound antibodies. No precipitin arc was obtained on immunodiffusion of antiserum and urine from the single patient investigated by this method. From the sensitivity of the indirect immunoassay it was concluded that the urine of the five patients contained less than 5% of the normal amount of cross-reacting material.