Susan Richards

Recombinant human acid α-glucosidase: Major clinical benefits in infantile-onset Pompe disease

Background: Pompe disease is a progressive metabolic neuromuscular disorder resulting from deficiency of lysosomal acid α-glucosidase (GAA). Infantile-onset Pompe disease is characterized by cardiomyopathy, respiratory and skeletal muscle weakness, and early death. The safety and efficacy of recombinant human (rh) GAA were evaluated in 18 patients with rapidly progressing infantile-onset Pompe disease. Methods: Patients were diagnosed at 6 months of age and younger and exhibited severe GAA deficiency and cardiomyopathy. Patients received IV infusions of rhGAA at 20 mg/kg (n = 9) or 40 mg/kg (n = 9) every other week. Analyses were performed 52 weeks after the last patient was randomized to treatment. Results: All patients (100%) survived to 18 months of age. A Cox proportional hazards analysis demonstrated that treatment reduced the risk of death by 99%, reduced the risk of death or invasive ventilation by 92%, and reduced the risk of death or any type of ventilation by 88%, as compared to an untreated historical control group. There was no clear advantage of the 40-mg/kg dose with regard to efficacy. Eleven of the 18 patients experienced 164 infusion-associated reactions; all were mild or moderate in intensity. Conclusions: Recombinant human acid α-glucosidase is safe and effective for treatment of infantile-onset Pompe disease. Eleven patients experienced adverse events related to treatment, but none discontinued. The young age at which these patients initiated therapy may have contributed to their improved response compared to previous trials with recombinant human acid α-glucosidase in which patients were older.

Early Treatment With Alglucosidase Alfa Prolongs Long-Term Survival of Infants With Pompe Disease

In a previous 52-wk trial, treatment with alglucosidase alfa markedly improved cardiomyopathy, ventilatory function, and overall survival among 18 children <7 mo old with infantile-onset Pompe disease. Sixteen of the 18 patients enrolled in an extension study, where they continued to receive alglucosidase alfa at either 20 mg/kg biweekly (n = 8) or 40 mg/kg biweekly (n = 8), for up to a total of 3 y. These children continued to exhibit the benefits of alglucosidase alfa at the age of 36 mo. Cox regression analyses showed that over the entire study period, alglucosidase alfa treatment reduced the risk of death by 95%, reduced the risk of invasive ventilation or death by 91%, and reduced the risk of any type of ventilation or death by 87%, compared with an untreated historical control group. Cardiomyopathy continued to improve and 11 patients learned and sustained substantial motor skills. No significant differences in either safety or efficacy parameters were observed between the 20 and 40 mg/kg biweekly doses. Overall, long-term alglucosidase alfa treatment markedly extended survival as well as ventilation-free survival and improved cardiomyopathy.

Recombinant Human Thyroid Stimulating Hormone: Development of a Biotechnology Product for Detection of Metastatic Lesions of Thyroid Carcinoma

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH). rhTSH was expressed by co-transfecting a human α-subunit cDNA with a human β-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be >99% by SDS polyacryl-amide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

Immune tolerance induction to enzyme-replacement therapy by co-administration of short-term, low-dose methotrexate in a murine Pompe disease model.

Clinical investigations of recombinant human acid α‐glucosidase for the treatment of Pompe disease often reveal the appearance of therapy‐specific antibodies. These antibodies could potentially interfere with recombinant human acid α‐glucosidase efficacy and induce immunological consequences. Several immunosuppressive agents, including methotrexate, mycophenolate mofetil and cyclosporin A with azathioprine, were evaluated for their potential to induce immune tolerance to recombinant human acid α‐glucosidase. Methotrexate was the only agent that reduced recombinant human acid α‐glucosidase‐specific antibody responses in acid α‐glucosidase knock‐out mice. A 3‐week, low‐dose methotrexate regimen controlled recombinant human acid α‐glucosidase‐specific antibody levels throughout 8u2003months of weekly recombinant human acid α‐glucosidase treatment. The success of this methotrexate regimen appears to require methotrexate administration within the first 24u2003h of recombinant human acid α‐glucosidase treatment. In an attempt to understand the benefit of methotrexate within the first day of recombinant human acid α‐glucosidase administration, the immune response 24u2003h following intravenous recombinant human acid α‐glucosidase treatment was investigated. A consistent expansion of peritoneal B1 B cells was observed. Control over this B1 B cell response may be part of the complex mechanism of action of methotrexate‐induced immune tolerance.

Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics

The administration of biological therapeutics may result in the development of anti-drug antibodies (ADAs) in treated subjects. In some cases, ADA responses may result in the loss of therapeutic efficacy due to the formation of neutralizing ADAs (NAbs). An important characteristic of anti-drug NAbs is their direct inhibitory effect on the pharmacological activity of the therapeutic. Neutralizing antibody responses are of particular concern for biologic products with an endogenous homolog whose activity can be potentially dampened or completely inhibited by the NAbs leading to an autoimmune-type deficiency syndrome. Therefore, it is important that ADAs are detected and characterized appropriately using sensitive and reliable methods. The design, development and optimization of cell-based assays used for detection of NAbs have been published previously by Gupta et al. 2007 [1]. This paper provides recommendations on best practices for the validation of cell-based NAb assay and suggested validation parameters based on the experience of the authors.

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.

Nephrotic Syndrome Complicating α-Glucosidase Replacement Therapy for Pompe Disease

We report a patient with Pompe disease who developed reversible nephrotic syndrome during prolonged, high-dose, experimental, enzyme replacement therapy with recombinant human acid α-glucosidase (rhGAA). Because of the development of antibodies to rhGAA and concomitant clinical decline, escalating doses of rhGAA were administered as part of an experimental immune tolerance regimen. Histologic evaluation of kidney tissue revealed glomerular deposition of immune complexes containing rhGAA itself, in a pattern of membranous nephropathy. To our knowledge, this is the first reported case of nephrotic syndrome occurring during enzyme replacement therapy. The nephrotic syndrome gradually resolved after the rhGAA dose was decreased, indicating that decreasing the antigenic load can ameliorate glomerular immune complex deposition associated with enzyme replacement in a highly sensitized patient.

Prolactin exerts hematopoietic growth-promoting effects in vivo and partially counteracts myelosuppression by azidothymidine

Prolactin (PRL) is a neuroendocrine hormone that influences immune and hematopoietic development. The mechanism of action of this hormone in vivo remains unclear; therefore, we assessed the effects of PRL on hematopoiesis in vivo and in vitro. Normal resting mice were treated with 0, 1, 10, or 100 microg of recombinant human prolactin (rhPRL) for 4 consecutive days and euthanized on the fifth day for analysis of myeloid and erythroid progenitors in the bone marrow and spleen. Both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL compared to the controls that had received saline only. Bone marrow cellularities were not significantly affected by any dose of rhPRL, but the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. These results suggest that rhPRL can promote hematopoiesis in vivo. Because rhPRL augments myeloid development in vivo, we examined the potential of the hormone to reverse the anemia and myelosuppression induced by azidothymidine (AZT). Mice were given rhPRL injections concurrent with 2.5 mg/mL AZT in drinking water. rhPRL partially restored hematocrits in the animals after 2 weeks of treatment and increased CFU-GM and BFU-e in both spleens and bone marrow. The experiments with AZT and rhPRL support the conclusion that the hormone increases myeloid and erythroid progenitor numbers in vivo, and they suggest that the hormone is clinically useful in reversing myelosuppression induced by AZT or other myeloablative therapies.

Minimal Effects on Immune Parameters Following Chronic Anti-TGF-β Monoclonal Antibody Administration to Normal Mice

Mice genetically deficient in TGF-β1 or TGF-β signaling capacity in T or B cells demonstrate profound immune dysregulation, as evidenced by increased lymph node size, expression of markers of memory/activation on T cells, inflammation in a variety of tissues and development of autoantibodies. However, this constant and complete lack of TGF-β1 or TGF-βR signaling may not reflect effects of TGF-β neutralization using antibodies in mature animals. Thus, the present studies were designed to determine if administration of an anti-TGF-β monoclonal antibody (neutralizes TGF-β1, 2 and 3) to mature, normal mice results in evidence of immune dysregulation or immune-mediated pathology. An initial study examined daily administration of 0.25, 0.75 and 2.5 mg/kg of anti-TGF-β to mice for three weeks, achieving blood levels of as high as 9 mg/ml. Comprehensive hematological and histopathological evaluation showed no evidence of pathology. A second study was designed to extend the antibody treatment period and further examine the functional status of the immune system. Mice were injected with 1 mg/mouse (approximately 50 mg/kg) of anti-TGF-β (1D11) three times per week achieving circulating blood levels of 1–2 mg/ml. Many parameters of immune status were assessed, including natural killer (NK) cell activity, lymphocyte proliferative responses, phagocytic activity, phenotypic assessment of leukocyte subsets, and serum measurements of proinflammatory cytokines, autoantibodies and immunoglobulin isotypes. In addition, histopathological assessment of heart, lungs, liver, kidney, salivary glands, skin, spleen and lymph nodes was also performed. Very few of the multiple immune parameters examined showed detectable changes in anti-TGF-β-treated mice. Changes that were observed were primarily restricted to the spleen and included increased spleen cell recoveries, increased percentages of macrophages, decreased percentages of NK cells, decreased phagocytic activity, decreased proliferative responses to mitogens and slight increases in T and B cells displaying an activated phenotype. Many of these same parameters examined in the lymph nodes were not altered by the anti-TGF-β treatment. The thymus was decreased in size, but altered only slightly in one population of developing T cells. Most of the changes observed were modest and returned to control levels after discontinuation of treatments. The only serological finding was an increase in IgA levels in anti-TGF-β-treated mice, but not in any other isotype. Finally, there was no evidence of increased inflammation in any of the peripheral tissues examined in the anti-TGF-β-treated mice. In conclusion, although there were changes in some of the immunological parameters examined in these studies, they were few and typically reversed following discontinuation of treatment. The modest nature of the changes observed in these studies is particularly evident when compared to published data of those same parameters examined in mice genetically deficient in TGF-β1 or mice having TGF-β unresponsive T or B cells. Thus, there does not appear to be any significant immune dysregulation detectable after long-term antibody-mediated neutralization of TGF-β in normal mice.

Use of human prolactin as a therapeutic protein to potentiate immunohematopoietic function

The conclusion that prolactin plays a role in immune and hematopoietic function was initially based upon observations in hormone deficient animals. The multiple cell function defects associated with hypophysectomy or bromocriptine treatment were reversed by administration of prolactin. Since these initial observations, an increasing body of literature supports prolactin having a role in the immune and hematopoietic system. A recombinant form of human prolactin (r-hPRL) has been produced and evaluated in a wide variety of preclinical models. Both in vitro and in vivo studies suggest that r-hPRL can enhance cell function, accelerate lymphoid and myeloid reconstitution and promote hematopoiesis. The multi-lineage effect of r-hPRL makes it an attractive candidate for clinical situations presenting with immune deficiency or myelosuppression.