Crystal Sung

A retrospective analysis of the potential impact of IgG antibodies to agalsidase β on efficacy during enzyme replacement therapy for Fabry disease

Fabry disease results from a genetic deficiency of alpha-galactosidase A (alpha GAL) and the impaired catabolism of globotriasoylceramide (GL-3) and other glycosphingolipid substrates, which then accumulate pathogenically within most cells. Enzyme replacement therapy (ERT) with agalsidase beta (Fabrazyme), one of two available forms of recombinant human alpha GAL, involves regular intravenous infusions of the therapeutic protein. Immunoglobulin G (IgG) antibodies to recombinant alpha GAL develop in the majority of patients upon repeated infusion. To explore whether anti-alpha GAL IgG interferes with therapeutic efficacy, retrospective analyses were conducted using data obtained from a total of 134 adult male and female patients with Fabry disease who were treated with agalsidase beta at 1mg/kg every 2 weeks for up to 5 years during placebo-controlled trials and the corresponding open-label extension studies. The analyses did not reveal a correlation between anti-alpha GAL IgG titers and the onset of clinical events or the rate of change in estimated GFR during treatment, and no statistically significant association was found between anti-alpha GAL IgG titers and abnormal elevations in plasma GL-3 during treatment. However, a statistically significant association was found between anti-alpha GAL IgG titers and observation of some GL-3 deposition in the dermal capillary endothelial cells of skin during treatment, suggesting that GL-3 clearance may be partially impaired in some patients with high antibody titers. Determination of the long-term impact of circulating anti-alpha GAL IgG antibodies on clinical outcomes will require continued monitoring, and serology testing is recommended as part of the routine care of Fabry disease patients during ERT.

High antibody titer in an adult with Pompe disease affects treatment with alglucosidase alfa

Clinical trials have demonstrated beneficial effects of enzyme replacement therapy (ERT) with alglucosidase alfa in infants, children and adults with Pompe disease. Recent studies have shown that high antibody titers can occur in patients receiving ERT and counteract the effect of treatment. This particularly occurs in those patients with classic-infantile Pompe disease that do not produce any endogenous acid α-glucosidase (CRIM-negative). It is still unclear to what extent antibody formation affects the outcome of ERT in adults with residual enzyme activity. We present the case of a patient with adult-onset Pompe disease. He was diagnosed at the age of 39years by enzymatic testing (10.7% residual activity in fibroblasts) and DNA analysis (genotype: c.-32-13T>G/p.Trp516X). Infusion-associated reactions occurred during ERT and the patients disease progressed. Concurrently, the antibody titer rose to a similarly high level as reported for some CRIM-negative patients with classic-infantile Pompe disease. Using newly developed immunologic-assays we could calculate that approximately 40% of the administered alglucosidase alfa was captured by circulating antibodies. Further, we could demonstrate that uptake of alglucosidase alfa by cultured fibroblasts was inhibited by admixture of the patients serum. This case demonstrates that also patients with an appreciable amount of properly folded and catalytically active endogenous acid α-glucosidase can develop antibodies against alglucosidase alfa that affect the response to ERT.

Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

BackgroundHLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patients immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.MethodsStandard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.ResultsThe validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.ConclusionOur results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses.

Pharmacokinetic and pharmacodynamic studies of two different rabbit antithymocyte globulin dosing regimens: results of a randomized trial.

Rabbit antithymocyte globulin (rATG; Thymoglobulin(®)) is currently used to prevent acute rejection in kidney transplantation. The dose and regimen of rATG have not been optimized. Moreover, the impact of different treatment regimens on T-cell phenotype reconstitution remains unknown. We conducted a prospective randomized study of 17 renal transplant patients to determine the pharmacokinetics of total and active (bound to human cells) rATG and T-cell phenotype reconstitution after rATG administration. Patients received rATG at a total dose of 6mg/kg, administered either as 1.5mg/kg/day on days 0-3 (Group 1, n=8) or 3mg/kg on days 0 and 3 (Group 2, n=9). All patients received tacrolimus, mycophenolate mofetil and steroids. Blood samples were assayed for total rATG by enzyme linked immunosorbent assay and active rATG by flow cytometry. Maximum concentrations and terminal half-lives were similar between the two groups but at month 3 Group 1 had significantly lower values for total rATG (concentration was 6.2±1.1μg/mL versus 10.2±2.9μg/mL in Group 2, p=0.027) and total rATG dose-normalized AUC (374±83dayg/mL versus 508±149dayg/mL in Group 2, p=0.046). Time to sub-therapeutic levels (<1μg/mL) of active rATG was significantly shorter in Group 1 (18.75±6.9days versus 20±7.5days in Group 2, p<0.001). rATG induced significant depletion followed by slow reconstitution of CD3(+), CD4(+) and CD8(+) cells, with no marked differences between groups. B-cell count was unaffected, whereas CD3(-)CD56(+) NK-cell depletion was observed in both groups. rATG induced a significant decrease in the proportion of naïve CD4(+) T-cells, which plateaued after month 1 in Group 1 and after month 6 in Group 2. The proportion of central memory CD4(+) T-cells increased to a similar extent in both groups (Group 1: 38±18% at baseline, 74±23% at one year, p=0.009; Group 2: 32±14% at baseline, 65±14% at one year, p=0.001). In conclusion, our results suggest that the dosing regimen for rATG induction influences pharmacokinetic parameters without affecting the quality of immune reconstitution.

Letter to the Editors: Concerning “CRIM-negative Pompe disease patients with satisfactory clinical outcomes on enzyme replacement therapy” by Al Khallaf et al

Dear Editors, In a recent paper in JIMD Reports, Al Khallaf et al. present two siblings (ages 4.5 and 2 years, respectively) with infantile Pompe disease (IPD). Despite being CRIM-negative [as determined by CRIM analysis using monoclonal antibodies specific for acid a-glucosidase (GAA)] and treated chronically with Myozyme® (alglucosidase alfa; rhGAA) enzyme replacement therapy (ERT), these patients had “unusually low anti-rhGAA antibody titers and good clinical outcome.” As the authors mention, the favorable outcomes seen in these children could potentially be due to the fact that they are, in fact, CRIM-positive as one of their mutations is a splice mutation. Though not detected by western blot, it is conceivable that sufficient enzyme protein is, nonetheless, detected by the immune system, which tolerizes to a significant extent. In addition, the authors speculate that the lower antibody titers seen in patient 2 could be due to her very early ERT initiation (at age 6 days) compared to her older brother (patient 1), who had commenced ERT at several months of age. Early initiation of ERT (defined here as ≤ 31 days) is important because of rapid disease progression in IPD. However, early commencement of ERT does not necessarily explain low(er) or no antibody formation, or necessarily preclude the possible need for immune tolerance induction (ITI), ceteris paribus. In two previous studies (Kishnani et al. 2010, Banugaria et al. 2011), some CRIM-negative and CRIM-positive patients developed high and sustained antibody titers despite early (age ≤ 31 days) initiation of ERT. This observation is further supported by data shown in Fig. 1 (Genzyme Corp.) for the 28 patients identified as ≤ 31 days old at the start of Myozyme® treatment (range: 1 to 31 days; mean: 17 days; median: 21 days). Two of 28 patients were CRIM-negative; information regarding CRIM status for the remaining patients was unavailable. In this cohort of 28 patients, 24 patients (86%) had seroconverted. The median peak titer for these 24 patients was 6,400. Five of 24 patients (21%; including the two known CRIM-negative patients) had peak titers ≥ 25,600 sustained for periods of time ranging from 3 months to > 1 year. One of the two documented CRIM-negative patients is also described in Abbott et al. (2011). This patient commenced ERT at day 10 of life [her parents had declined immune tolerance induction (ITI)] and had a peak titer of 25,600 at month 27 of ERT. The second CRIM-negative patient commenced ERT at age 2 weeks and had a peak titer of 409,600. Two of the 28 patients received immunomodulation (CRIM status unknown): one patient had a peak titer of 51,600, which persisted for 6 months post-peak before declining to 200 subsequent to immunomodulation; the second patient received ITI prophylactically and as of the last study time point had not seroconverted. Neutralizing antibody activity, including neutralization of enzyme uptake and catalytic activity, was tested in six of 28 patients. The single patient who tested positively (for inhibition of enzyme uptake) had a high sustained antibody titer of 409,600. While “early ERT initiation” in this analysis was considered as ERT commenced at or before 31 days of age, data from future studies [including those related to newborn screening (NBS)] could lead to a reconceptualization of what is conceived of as “early ERT initiation” and the timescale generally applied. Fig. 1 Peak antibody titers for patients ≤ 1 month of age upon initiation of enzyme replacement therapy with Myozyme®. In a case report by Rohrbach et al. (2010), the authors concluded that an IgE inhibitor, omalizumab, used to mitigate the allergic response, could have played an immunomodulatory role that limited the formation of anti-Myozyme® IgG antibodies in this patient. Based on findings from the subsequent study by Abbott et al. involving a CRIM-negative patient who had only moderately increased, yet persistent, titers (“atypical immune response”), as well as the two new cases from Al Khallaf et al. (none of the 3 patients represented in these two studies received ITI), it is evident that some patients designated as CRIM-negative do not develop high antibody titers with ERT. As we have previously discussed in Abbott et al. (2011), the persistence of titers (not just the presence of high titers per se) could potentially have important clinical implications. In the case by Abbott et al., the patient’s titers peaked at 25,600 after approximately two years of treatment. Titers then fluctuated between 12,800 and 25,600 over the subsequent six months. Although titers ultimately decreased to 6,400 the following year (without immunomodulation, and similar to titers seen for patient 1 in Al Khallaf et al.), her demise was associated with titers sustained in this 6,400 range. This contrasts with what has been seen in long-term infantile survivors, who at most recent follow-up (n=10; time on ERT 23–129 months), had titers of 0 to 1,600 (Prater et al. 2012). The potential clinical impact of titers that persist in low to moderate ranges has yet to be fully characterized and warrants further study. At present, there is limited information to accurately predict which patients with CRIM-negative IPD are unlikely to develop HSAT or very low titers with regular ERT administration (ERT initiated without ITI). Indeed, most CRIM-negative patients treated in this manner have developed HSAT and have had poor clinical outcomes (e.g., Banugaria et al. 2011). The risk-benefit ratio, therefore, supports the administration of prophylactic short-term ITI (relatively more safe) to CRIM-negative patients at the time of ERT initiation, as opposed to longer-term immune suppression (relatively less safe and less certain efficacy) once HSAT has developed (Messenger et al. 2012; Banugaria et al. 2011). The report by Al Khallaf et al. builds upon our knowledge that amongst patients designated as CRIM-negative who receive ERT (without ITI), there appears to be a small subset that does not develop high titers per se, but rather a more variable antibody response. However, it is important to remember that the vast majority of CRIM-negative patients (even those diagnosed early, e.g., age ≤ 31 days) do develop high and sustained titers. Consequently, at this time, the risk-benefit ratio justifies the use of ITI in all CRIM-negative patients at the start of ERT. We are continuing to assess the potential clinical impact of antibody titers (low to moderate titers) that remain sustained. Further work exploring the basis for variable immune responsiveness in such patients, including age at start of ERT, potential HLA associations, whether the CRIM assay may miss some CRIM-positive patients, and other genomic factors, is needed.

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

BACKGROUND Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. RESULTS We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. CONCLUSION The new approach greatly improves assay precision and accuracy.

Enhancement of human plasma glucosylceramide assay sensitivity using delipidized plasma.

Glucosylceramide (GL-1) level in human has been considered as a surrogate biomarker for enzyme replacement and substrate reduction therapies (ERT and SRT) for Gaucher and Fabry patients. Due to the high endogenous level of GL-1 in human plasma, it is difficult to achieve the analytical sensitivity of plasma GL-1 below the normal endogenous level (1.7 μg/mL to 6.6 μg/mL) when using the standard addition method and regular plasma matrix for standard curve. A high sensitivity plasma GL-1 assay with LLOQ at 0.1 μg/mL was developed and validated using delipidized plasma so that patient plasma concentrations that are below normal reference range can be measured accurately. The normal reference range was established from 120 healthy donors using this developed new method. Twenty-three Fabry patient plasma samples including baseline and post-investigation drug treatment samples were measured. All post-treatment samples showed GL-1 concentration below 2.0 μg/mL, indicating the utility of the reported high sensitivity assay using delipidized plasma for monitoring the plasma GL-1 biomarker level in patients.

An immune tolerance approach using transient low-dose methotrexate in the ERT-naïve setting of patients treated with a therapeutic protein: experience in infantile-onset Pompe disease.

PurposeTo investigate immune tolerance induction with transient low-dose methotrexate (TLD-MTX) initiated with recombinant human acid α-glucosidase (rhGAA), in treatment-naïve cross-reactive immunologic material (CRIM)-positive infantile-onset Pompe disease (IOPD) patients.MethodsNewly diagnosed IOPD patients received subcutaneous or oral 0.4 mg/kg TLD-MTX for 3 cycles (3 doses/cycle) with the first 3 rhGAA infusions. Anti-rhGAA IgG titers, classified as high-sustained (HSAT; ≥51,200, ≥2 times after 6 months), sustained intermediate (SIT; ≥12,800 and <51,200 within 12 months), or low (LT; ≤6400 within 12 months), were compared with those of 37 CRIM-positive IOPD historic comparators receiving rhGAA alone.ResultsFourteen IOPD TLD-MTX recipients at the median age of 3.8 months (range, 0.7–13.5 months) had a median last titer of 150 (range, 0–51,200) at median rhGAA duration ~83 weeks (range, 36–122 weeks). One IOPD patient (7.1%) developed titers in the SIT range and one patient (7.1%) developed titers in the HSAT range. Twelve of the 14 patients (85.7%) that received TLD-MTX remained LT, versus 5/37 HSAT (peak 51,200–409,600), 7/37 SIT (12,800–51,000), and 23/37 LT (200–12,800) among comparators.ConclusionResults of TLD-MTX coinitiated with rhGAA are encouraging and merit a larger longitudinal study.