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Clinical development of Enzyme Replacement Therapy in alpha-Mannosidosis patients using recombinant human enzyme

Final Report Summary - ALPHA-MAN (Clinical development of Enzyme Replacement Therapy in alpha-Mannosidosis patients using recombinant human enzyme.)

Executive Summary:
The lysosomal storage disorder (LSD) alpha-Mannosidosis is a rare genetic disease and according to the EU regulations, designated as an “orphan“ disease. Alpha-Mannosidosis is caused by an enzyme defect due to mutations in the gene for lysosomal alpha-Mannosidase (LAMAN) affecting the lysosomal and cellular glycoprotein catabolism with severe consequences for the organism. In humans, LAMAN deficiency results in progressive mental retardation, skeletal changes, hearing loss and recurrent infections and many patients die during early childhood. Today, the most promising therapy for lysosomal storage disorders including alpha-Mannosidosis is Enzyme Replacement Therapy (ERT) where the respective enzyme lacking in the patient is produced by recombinant approaches and then introduced into the blood stream, from where it is internalized by the cells and reaches the lysosomes replacing the missing endogenous enzyme. To date, no specific treatment for alpha-Mannosidosis is available. Since children are typically born clinically healthy, an early initiated therapy shortly after birth could dramatically improve their life expectancy and quality of life.
Since pharmaceutical interest in this disease is low, two EU-supported projects (EURAMAN and HUE-MAN) within the 5th and 6th framework program, respectively have worked towards developing the recombinant human enzyme (rhLAMAN) as a therapeutic agent for patients suffering from alpha-Mannosidosis. The promising results of these two previous networks in general, but especially the achievements of the HUE-MAN project, including i) the large scale production of the recombinant human enzyme, ii) the evaluation of disease progression in alpha-Mannosidosis patients, iii) the determination of clinical endpoints through the natural history study and iiii) the development of an effective ERT protocol in pre-clinical mouse studies, were the basis for the ALPHA-MAN project within the 7th framework program. In addition, but independent of HUE-MAN, to demonstrate the safety of the product, toxicology studies have been completed. The main objectives of the ALPHA-MAN network was to transfer and expand the information and knowledge gained from the previous EURAMAN and HUE-MAN projects, to enable us to perform “First in Man” clinical trials in alpha-Mannosidosis patients, using the medicinal enzyme product rhLAMAN as a therapeutic agent and furthermore to improve the knowledge regarding i) long term “chronic dosing” including the determination of the minimal effective dose in the mouse model and ii) mechanism of lysosomal enzyme transfer across the Blood Brain Barrier (BBB), in a newly established immune-tolerant mouse model. The final goal of ALPHA-MAN was to make a future treatment for ALL alpha-Mannosidosis patients available and thereby dramatically improving their life expectancy and quality of life.
In line with our plans we have been able to demonstrate the safety and clinical efficacy of rhLAMAN as an effective therapeutic agent for treatment of the human disease alpha-Mannosidosis in clinical trial Phases 1 and 2. Within the clinical trials, the dose levels based on the results in the non-clinical tests, were confirmed and a minimum effective dose in humans established. A clinical phase 3 study was initiated and will be finalized by June 2014. In non-clinical studies we also found the optimal rhLAMAN dose for a maximal correction of visceral and central nervous system pathology following injection in immune-tolerant alpha Mannosidosis mice. Putative receptors that are responsible for rhLAMAN uptake into the brain across the Blood Brain Barrier were investigated and we were able to study the outcome of long-term ERT on the neuropathology and associated behavioural deficits observed in immune-tolerant alpha-Mannosidosis mice.

Project Context and Objectives:
The LSD alpha-Mannosidosis belongs to the glycoproteinoses affecting the glycoprotein catabolism and is characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual disability. Alpha-Mannosidosis occurs in approximately 1 of 500,000 live births [1] and is expected to be found in any ethnic group anywhere in the world. Alpha-Mannosidosis is caused by mutations in the MAN2B1 (LAMAN) gene encoding lysosomal alpha-Mannosidase (LAMAN). LAMAN is a lysosomal hydrolase that is responsible for cleaving alpha-mannosidic linkages during the ordered degradation of oligosaccharides. Only after degradation, the sugars can leave the lysosomes. In alpha-Mannosidosis, the deficiency of alpha-Mannosidase activity causes a block in the degradation of glycoproteins, leading to an intralysosomal accumulation of mannosyl linked oligosaccharides, that can be detected in all tissues as well as in urine and serum of patients. The progressive accumulation of oligosaccharides is somehow toxic to the cells, some types of cells are more sensitive than others [2-4]. The children are often born apparently normal, and their condition worsens progressively. Since children are born clinically healthy and due to the progressive character of the disease, an early diagnosis and early initiated Enzyme Replacement Therapy could prevent the fatal outcome of this disease.

The clinical findings in alpha-Mannosidosis include a broad range of symptoms from an early lethal form to less symptomatic forms, initially diagnosed in children [1]. Like all other LSDs, alpha-Mannosidosis lacks a genotype to phenotype correlation and even among siblings carrying the same mutation, clinical variations occur.
Intravenous administration of the missing enzyme has been shown in several animal models of lysosomal storage disorders to effectively reduce the lysosomal storage in multiple visceral tissues. In the non-neuronopathic type I Gaucher disease which primarily affects cells of the monocyte/ macrophage lineage, ERT with a modified form of the missing glucocerebrosidase that targets mannose receptors in monocytes/macrophage has become a powerful therapeutic option. A major problem for ERT of most of the lysosomal storage disorders is the involvement of the central nervous system. The blood–brain barrier apparently prevents the crossing of lysosomal enzymes from the blood to the interstitial space surrounding neuronal and glial cells of the central nervous system. In spite of the inaccessibility of neural cells to intravenously administered lysosomal enzyme, ERT may have some indirect beneficial effects on brain function in lysosomal storage disorders. The lysosomal damage may result from the clearance of storage material from brain endothelia and from the meninges, which may improve the microcirculation and the flow of the cerebrospinal fluid. The assessment of the efficacy of ERT depends on the availability of a suitable animal model and of sufficient amounts of the missing enzyme. The generation of a mouse model for α-Mannosidosis and the production of the recombinant LAMAN have made it possible to study the efficacy of ERT in this lysosomal storage disorder.
To study the pathology and therapeutic approaches of the carbohydate storage disorder alpha-Mannosidosis, several animal models including mice, cats and guinea pigs have been evaluated. Alpha-Mannosidosis mice have been generated by targeted disruption of the mouse LAMAN gene (LAMAN KO) [5]. These mice have been shown to be a valid model for alpha-Mannosidosis [6, 7] reflecting a mild form, when compared to the human disease, whereas the naturally occuring alpha-Mannosidosis cats [8] and guinea pigs [9-12] display a more severe form, with decreased life spans and severe neurological features, such as demyelination, neurodegeneration and inflammation. However, within HUE-MAN, detailed biochemical and morphological analyses revealed a distinct and specific neuropathology in the cerebellum of alpha-Mannosidosis mice, that has not been described previously. The observation of the neuropathological abnormalities is of great use, since it can now be used as a “clinical” endpoint to study the efficacy of ERT in brains of alpha-Mannosidosis mice.
Therapeutic correction of storage lesions after LAMAN enzyme replacement in the preclinical mouse model in the central nervous system (CNS) could be demonstrated. The correction of lysosomal storage in the CNS worked when higher doses of enzyme (>250U/kg) were injected frequently (at least 4x) in short intervals (3.5 days). Clearance of storage was observed in neurons of the CNS and peripheral nervous system (PNS). An uptake of the recombinant enzyme could be demonstrated in hippocampal neurons of the CNS. The morphological correction in brain was also accompanied by an improvement motor coordination.
The disadvantage using alpha-Mannosidosis mice for ERT is, that they develop a severe immune-reaction after frequent injections of the human LAMAN enzyme precluding long-term ERT. Since chronic dosing studies will help us to find i) the mechanism by which the enzyme crosses the Blood Brain Barrier (BBB) and ii) to see, to which extent, the observed neuropathology in alpha-Mannosidosis mice is reversible, an immune-tolerant alpha-Mannosidosis mouse model was generated within the HUE-MAN project, that will allow chronic dosing with the human enzyme. The idea behind this is, that a mouse model, that is deficient for the mouse LAMAN, but expresses an inactive form of the human enzyme, shows the “classical” KO phenotype, but tolerates the injected human enzyme, allowing chronic ERT studies. This approach has already been successfully used to generate an immune-tolerant mouse model for the LSD “Metachromatic Leukodystrophy” [13]. To date, no real treatment for alpha-Mannosidosis is available. Since children are born healthy, an early initiated therapy shortly after birth could dramatically improve their life expectancy and quality of life. Since pharmaceutical interest in this disease is low, two EU sponsored projects (EURAMAN and HUE-MAN) within the 5th and 6th framework program, respectively have worked towards developing the recombinant human enzyme (rhLAMAN) as a therapeutic agent for patients suffering from alpha-Mannosidosis and are now the base for clinical trials in alpha-Mannosidosis. The enzyme has received Orphan Drug Designation in January 2005 (EU/3/04/260). The major achievements of these two collaborative projects are listed below:
Within the 5th EU framework the consortium EURAMAN (2001-2005) successfully established:

1. Development of simple diagnostic tests (PCR or microchips based) for all disease causing mutations
2. Spectrum of European mutations in the alpha-Mannosidase gene (MAN2B1)
3. Biochemical characterization of the consequences of alpha-Mannosidosis mutations
4. Phenotype/Genotype correlation
5. Industrial production of three different therapeutic agents for alpha-Mannosidosis
6. Preclinical studies on the effect of the three therapeutic enzymes on a mouse model of alpha-Mannosidosis
7. Increased understanding of the oligomannoside metabolism and its importance in pathology
8. Detailed characterization of the behavioural phenotype of alpha-Mannosidosis mice

Within the 6th EU framework the collaboration HUE-MAN (2006-2009) successfully established:
1. Large scale production of recombinant human LAMAN as a therapeutic agent for Enzyme Replacement Therapy (ERT) in alpha-Mannosidosis
2. Determination of the most effective dose in preclinical trials using rhLAMAN in alpha-Mannosidosis mice
3. Generation of an immune-tolerant mouse model that will allow chronic ERT treatment
4. Natural history study of 45 alpha-Mannosidosis patients
5. Determination of clinical endpoints for future clinical trials
6. Collection of patient and mutational data in a database accessible to the public

References

1. Malm, D. and O. Nilssen, Alpha-mannosidosis. Orphanet J Rare Dis, 2008. 3: p. 21.
2. Heikinheimo, P., et al., The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation. J Mol Biol, 2003. 327(3): p. 631-44.
3. Jolly, R.D. et al., Mannosidosis: patterns of storage and urinary excretion of oligosaccharides in the bovine model. Aust J Exp Biol Med Sci, 1980. 58(4): p. 421-8.
4. Wong, L.T. et al., Oral zinc therapy in the treatment of alpha-mannosidosis. Am J Med Genet, 1993. 46(4): p. 410-4.
5. Stinchi, S., et al., Targeted disruption of the lysosomal alpha-mannosidase gene results in mice resembling a mild form of human alpha-mannosidosis. Hum Mol Genet, 1999. 8(8): p. 1365-72.
6. Caeyenberghs, K., et al., Multivariate neurocognitive and emotional profile of a mannosidosis murine model for therapy assessment. Neurobiol Dis, 2006. 23(2): p. 422-32.
7. D'Hooge, R., et al., Neurocognitive and psychotiform behavioral alterations and enhanced hippocampal long-term potentiation in transgenic mice displaying neuropathological features of human alpha-mannosidosis. J Neurosci, 2005. 25(28): p. 6539-49.
8. Goodman, L.A. P.O. Livingston, and S.U. Walkley, Ectopic dendrites occur only on cortical pyramidal cells containing elevated GM2 ganglioside in alpha-mannosidosis. Proc Natl Acad Sci U S A, 1991. 88(24): p. 11330-4.
9. Crawley, A.C. et al., Alpha-mannosidosis in the guinea pig: a new animal model for lysosomal storage disorders. Pediatr Res, 1999. 46(5): p. 501-9.
10. Crawley, A.C. et al., Enzyme replacement therapy in alpha-mannosidosis guinea-pigs. Mol Genet Metab, 2006. 89(1-2): p. 48-57.
11. Crawley, A.C. and S.U. Walkley, Developmental analysis of CNS pathology in the lysosomal storage disease alpha-mannosidosis. J Neuropathol Exp Neurol, 2007. 66(8): p. 687-97.
12. Robinson, A.J. et al., Behavioural characterisation of the alpha-mannosidosis guinea pig. Behav Brain Res, 2008. 186(2): p. 176-84.
13. Matzner, U., et al., Induction of tolerance to human arylsulfatase A in a mouse model of metachromatic leukodystrophy. Mol Med, 2007. 13(9-10): p. 471-9.

The main objectives of the ALPHA-MAN network were to transfer and expand the information and knowledge gained from the many years of work from the previous EURAMAN and HUE-MAN projects, to enable to perform “First in Man” clinical trials in alpha-Mannosidosis patients, using the medicinal enzyme product rhLAMAN as the therapeutic agent and furthermore to improve the knowledge regarding i) long term “chronic dosing” and ii) mechanism of lysosomal enzyme transfer across the Blood Brain Barrier, in a newly established immune-tolerant mouse model. The final goal of ALPHA-MAN was to make a future treatment for ALL alpha-Mannosidosis patients available and thereby dramatically improve their life expectancy and quality of life. In addition, the activities of ALPHA-MAN will increase the knowledge about the mechanism of how lysosomal enzymes can cross the Blood Brain Barrier, which is also of great medical importance for the treatment of other neurodegenerative disorders.
Participants in this European network include clinicians, academics and industry (Small and Medium sized Enterprises - SME) and most of them have already participated in the successful networks EURAMAN and HUE-MAN, in which the medicinal product and pre-clinical studies have been developed. The combination of top-level European scientists and experienced clinicians facilitate the integration of research capacities across Europe, increasing coherence and providing critical mass of investigators. The integrated multidisciplinary research enables direct interactions between technology and biology and will provide the knowledge base essential for the rational design of therapeutic interventions. The unique composition and collection of expertise of this consortium was of major importance to pave the way for a successful introduction of ERT for human patients suffering from Alpha-Mannosidosis. To achieve the general goal, the project has been divided into four objectives:

1) Implementation of clinical trials in alpha-Mannosidosis patients to provide an effective drug for the orphan disease alpha-Mannosidosis.
➢ The aim of this study was to perform clinical trial Phases 1, 2 and 3 in patients to demonstrate the safety and clinical efficacy of rhLAMAN as an effective therapeutic agent for treatment of the human disease alpha-Mannosidosis.
2) Determination of the minimal effective dose by chronic treatment studies in immune-tolerant alpha-Mannosidosis mice.
➢ The aim of this study was to find the minimal effective dose for a maximal correction of visceral and central nervous system pathology.
➢ Immune-tolerant alpha Mannosidosis mice should be chronically injected once a week with different doses of rhLAMAN and analyzed for their storage in visceral and central nervous tissues.
3) Studies of the mechanism how recombinant human LAMAN crosses the Blood Brain Barrier.
➢ The aim of this study was to find putative receptors that are responsible for rhLAMAN uptake into the brain across the Blood Brain Barrier.
➢ Immune-tolerant alpha-Mannosidosis mice should be injected with high doses rhLAMAN and analyzed for binding partners by various biochemical methods.
4) Studies of the impact on chronic ERT treatment on the neuropathology and underlying behavioural deficits in alpha-Mannosidosis mice.
➢ The aim of this study was to see whether long-term ERT treatment can prevent and/or reverse the neuropathology and associated behavioural deficits observed in immune-tolerant alpha-Mannosidosis mice.
➢ Immune-tolerant alpha-Mannosidosis mice should be chronically injected with the minimal effective dose (see Objective 2) and analysed for their neuropathology by either immuno-histological/biochemical or behavioral assays.

Project Results:
The present project was of great importance for the introduction of recombinant human lysosomal alpha-mannosidase as an ERT in patients. In line with our plans we have been able to demonstrate clinical efficacy and safety of rhLAMAN as an effective therapeutic agent for treatment of the human disease alpha-Mannosidosis in clinical trial Phases 1, 2 and 3, respectively. Within the clinical trials, the dose levels obtained from the results in the pre-clinical tests and the minimum effective dose established in clinical trials, were used in the pivotal studies. In pre-clinical studies we also found the optimal rhLAMAN dose for a maximal correction of visceral and central nervous system pathology following injection in immune-tolerant alpha Mannosidosis mice. We have analyzed putative receptors that are responsible for rhLAMAN uptake into the brain across the Blood Brain Barrier and we were able to study the outcome of long-term ERT on the neuropathology and associated behavioural deficits observed in immune-tolerant alpha-Mannosidosis mice.

The major achievements of the project include:

1. It could be demonstrated that the newly generated immune-tolerant alpha-Mannosidosis mice are a valid mouse model for alpha-Mannosidosis reflecting the human disease to the same degree as the classical alpha-Mannosidase Knockout mice. First long term treatments using this mouse model revealed that they are immune-tolerant against the injected recombinant human enzyme (Lamazym). Biweekly and weekly injections of doses between 125 and 750U/kg over a period of 12 weeks revealed efficient storage reduction upon use of all doses in the CNS. In visceral organs complete storage reduction was mostly observed already when using the lowest dose of 125U/kg. However, in brain the use of 500U/Kg and 750U/kg dose was always shown to be most effective in storage reduction as well as in regard to normalization of parameters that appear secondary to the primary substrate storage. Therefore we have chosen biweekly injections of 500U/kg as the CNS minimal effective dose that in mice will be used in future studies. It was revealed that the oligosaccharide storage phenotype in central nervous and visceral tissues was comparable to the classical knockout mice. Similarily, neuropathological changes that occur secondary to the primary storage in brains such as inflammation and lysosomal dysfunction were observed. Brains of mice that were treated over a period of 14 weeks and longer with the minimal effective dose (500U/kg) and doses below (125U/kg) showed a dosage dependent i) uptake of LAMAZYM into the brain and ii) reduction of primary sugar storage which was more than 50% upon use of the highest dose. This was reflected by a reversal of the lysosomal pathology and reduction of microglia. On a functional level long-term ERT improved treadmill performance, exploratory activities, spatial learning and memory of alpha-mannosidosis mice. A rescue in defective hippocampal long-term potentiation measured after ERT could explain the beneficial effects on spatial learning in these mice.

2. Clinical Phase Studies 1 to 3 were prepared and monitored. The Pre-Trial regulatory filings for the IMPD were prepared and submitted as part of the authority evaluation af all clinical trials. The Phase 2a protocol, including a synopsis, and Patient information /Consent forms were prepared and submitted. The application was approved by DKMA (Danish Medicine Agency). Amendments A1, A2, A3, A4 and A5 were submitted for the Phase 2a, and this includes that the trial was prolonged from 6mths to 13 mths duration of treatment. The Phase 2a was conducted, monitored and completed, and the Authorities were supplied with the Declaration of End of Trial. The Phase 2b protocol, including a synopsis, and Patient information/consent forms were prepared and submitted to EU Voluntary Harmonized Procedure (VHP) as the trial is a multinational EU trial. After VHP evaluation the application was submitted to each national country authority. The DKMA, BfArM, MHRA, ANSM, FAGG and EAMPS approved the application. Amendments A1 and A2 were submitted for Phase 2b, and this includes that the trial was prolonged from 6mths to 16 mths duration of treatment. The Phase 2b was conducted, monitored and completed, and the Authorities were supplied with the Declaration of End of Trial. The Phase 3 protocol, including a synopsis, and Patient information/consent form were prepared and submitted to several national country authorities, as the trial is multinational. The DKMA, BfArM, MHRA, ANSM, FAGG, EAMPS and MPA approved the application. Amendments A1, A2, A3 and A4 were submitted for Phase 3, and this includes that the number of trial subjects was expanded from 20 to 25 patients. An independent Data Monitoring Committee was established and evaluated the blinded interim data in the Phase 3 trial. The Phase 3 was conducted (though at writing still ongoing) and monitored.

3. The Phase 1 trial was conducted, monitored and completed without safety concerns. This represented the first-in-man clinical trial with enzyme replacement therapy in alpha-Mannosidosis patients. This was a major achievement for patients with alpha-Mannosidosis as well as a prerequisite for the conductance of the following clinical phases with a focus on clinical effectiveness. Also very interesting data concerning the pharmaco-kinetics of rh-LAMAN were generated, which included a much longer half-life and thus a longer presence of rh-LAMAN in the blood as compared with other enzymes developed for enzyme replacement therapy in lysosomal storage disorders.


4. The Phase 2a and 2b clinical trials were performed with all patients evaluated in Copenhagen. There was a very high degree of compliance (99%), with completion of almost all infusions. This is a major achievement in this logistically very demanding and long study. One very important achievement was the documentation, that Lamazym treatment was associated with a statistically significant improvement for a number of the biochemical and clinical efficacy endpoints. From a safety point of view, Lamazym was well tolerated in the patients. The data suggests that ERT with Lamazym is an encouraging new treatment for patients with alpha-Mannosidosis. Both Phase 2a and 2b were prolonged to ensure that safety and efficacy evaluation were performed on the best possible background.

5. For the Phase 3 study it was decided after discussion with the European Medicines Agency (EMA) to change the placebo:active ratio from 33% to 40%, and to change the duration of the study from 6mth to 12 mths. In the Phase 3 protocol treatment with either placebo or a minimum effective dose of 1 mg active Lamazym per kg body weight, was given as an intravenous infusion once weekly. The trial was performed with 25 European patients from Norway, Sweden, UK, Denmark, Netherland, Belgium, Polen, Germany, France and Spain. All evaluations were performed in Copenhagen. Being a placebo controlled trial, it is a great achievement to have been able to include 25 patients from this ultra rare disease and that compliance of completion of infusion and visits have been >98%.

6. Since April 2012, twenty seven additional patients have been registered and subjected to mutation analysis. The mutation database (amamutdb.no) now includes more than 240 patients from 42 countries and describes 166 different mutations of which 150 are pathogenic. Data on enzyme activity, intracellular transport, processing and effect on 3D-folding have been generated for 48 missense variants. The genotypes of all patients included in the ALPHA-MAN clinical trials have been determined and the molecular features of the corresponding mutant gene products have been evaluated. Two editions of the database have been developed; a public internet version in which sensitive patient information and unpublished data have been omitted, and a pass-word protected version which holds additional unpublished data and patient sensitive information. T Clinical features, as reported by responsible physicians through questionnaires, have been re-evaluated, systemized, and re-deployed, as well as being published as a separate article.

7. A method was succesfully developed to screen high-mannose type oligosaccharides present in plasma, urine, and cerebrospinal fluid of patients suffering from alpha Mannosidosis. This method has been applied to quantify especially Mannose-2 in these body fluids from patients treated with ERT. The results demonstrated that the ERT significantly lowered the Mannose-2 in the body fluids from patients treated with ERT in clinical trial phase 2 and 3.

8. The different data obtained in the clinical trials were successfully managed in a clinical Data Management System. All clinical data that have been collected during the clinical trials were evaluated with completion of the clinical trials. Industry level databases have been defined for the trials, data have been retrieved, validated and analysed statistically and integrated clinical reports have been finalised for the Phase 1, 2A, 2B and 3 trials.


9. We have organised effective research training, meetings and communication events between laboratories and clinical centers. This has greatly stimulated the collaborative network and facilitated the achievement of the research goals of the ALPHA-MAN project.

10. In part due to the success of the clinical trials the SME partner 2 (Zymenex) was taken over by Chiesi-Pharmaceutical (August 2013)

11. The dissemination of knowledge was most prominently expressed by a number of press releases, publications, communications and presentations at international conferences and approvals by different authorities. Project management and ALPHA-MAN consortium meetings have been held every ½ year. In addition regular phone conferences and small meetings with selected participants from the clinical partners and the basic science partners of ALPHA-MAN were held. The project website was maintained and regularly updated. The participants of the ALPHA-MAN project have given presentations at international congresses and published articles which have highlighted the importance of the ALPHA-MAN network to experts but also to the general public. The ALPHA-MAN project has been active in research training, exemplified by visits of the scientists and clinicians among the ALPHA-MAN laboratories and clinical centers. The midterm, second periodic report and final report and corresponding cost statements have been prepared.

12. The management has been able to foster several fruitful collaborations amongst ALPHA-MAN scientists and clinicians. The project website provides all the information of the ALPHA-MAN network events and was regularly used as a contact site for patients and relatives. An important task of the management was the organization of consortium meetings. An amendment request, reports, work plans and cost statements have been submitted to the EC. Public participation and awareness were raised through a number of articles and presentations in newspapers and journals, respectively.

Preclinical studies (WP2):

The aim was a biochemical, histological and behavioral characterization of the immune-tolerant mouse model as well as to establish a long term protocol with a minimal effective dose for storage reduction in the central nervous system (CNS). Novel immune-tolerant α-Mannosidosis knockout mice were investigated to determine their validity to serve as a genuine alpha-Mannosidosis mouse model. Therefore we started on the one hand a detailed characterization of the novel immune-tolerant mouse model for transgene expression, immunetolerance, sugar storage and neuropathology. On the other hand we initiated a behavioral comparison of the immune-tolerant mouse model and the original mouse model that was used during the previous HUE-MAN project. In addition we have performed first long term ERT over a period of 12 weeks by injecting different doses (125-750U/kg) in different intervals (weekly vs. weekly) to find the minimum effective dose for an effective treatment of CNS storage and storage related neuropathology. In terms of behavioral analyses we selected tests based upon previous research in these mice and included the assessment of visuo-spatial learning (Morris Water Maze), emotional function and exploration (Open Field), and ambulatory abilities (Treadmill). Furthermore, we evaluated the substitutability of wildtype mice with heterozygous mice to function as control group, which would allow for more effective breeding from an economical and ethical perspective. Now we are in the progress of the first long-term enzyme replacement therapy (ERT) trial in these mice that will be behaviorally evaluated. Mice (age 3 months – early symptomatic stage for alpha-Mannosidosis mice) are receiving bi-weekly intravenous injections of 500U/kg rhLAMAN for 14 weeks. During the last 2 weeks of the treatment, functional efficacy will be assessed by the aforementioned behavioral protocols. Immune-tolerant knockout mice tolerate chronic administration of the industrial produced recombinant enzyme and first long term ERTs with different doses have been performed and the effect on the observed visceral and neuronal pathology have been evaluated. By this approach the use of 500U/kg has been established as the CNS most effective dosage. Based on this we have performed different long term ERTs (maximal duration of 6 months) and analyzed the effect of ERT on the observed neuropathology and underlying behavioral deficits.
Behavioral criteria for therapeutic evaluation were successfully confirmed for the novel immune-tolerant alpha-Mannosidosis mice during the first reporting period. During the second reporting period, we evaluated the behavioral efficacy of long-term ERT in different studies. Immune-tolerant alpha-Mannosidosis mice (age of initiation ~ 3 months) received bi-weekly intravenous injections of 500U/kg rhLAMAN for 14 weeks. During the last weeks of treatment functional efficacy was assessed by the following behavioral tests: Morris Water Maze (learning & memory), Open Field (emotional function & exploration) and Treadmill (motor performance). In another study, immune-tolerant alpha-Mannosidosis mice (age of initiation ~ 2 months) received weekly injections of 500U/kg rhLAMAN. These mice were treated for ~ 9 months in total. They received a first behavioral evaluation at the age of ~ 4.5 months. A second behavioral evaluation was performed at the age of ~ 8.5 months. Finally these mice also underwent electrophysiological evaluation (long-term potentiation) at the age ~ 10.5 months. For all behavioral experiments, these mice were compared to alpha-Mannosidosis and control mice receiving similar treatment regimens of intravenous saline.
It could be shown that immune-tolerant alpha-Mannosidosis mice are a valid mouse model for alpha-Mannosidosis reflecting the human disease to the same degree as the classical alpha-Mannosidase Knockout mice. We have also established effective long term treatments over a period of 12 weeks using the immune-tolerant mice that have been biochemically proven to be immune-tolerant against the injected recombinant human enzyme (rhLAMAN). In contrast to the classical alpha-Mannosidase knockout mice that die upon frequent injections of LAMAZYM due to severe humoral responses the immune-tolerant mice did not develop antibodies against the injected enzyme and did not show any signs of immunological reactions. Biweekly and weekly injections of doses between 125 and 750U/kg over a period of 12 weeks revealed efficient storage reduction upon use of all doses in the CNS. In visceral organs complete storage reduction was mostly observed already when using the lowest dose of 125U/kg. However, in brain the use of 500U/Kg and 750U/kg dose was always shown to be most effective in storage reduction as well as in regard to normalization of parameters that appear secondary to the primary substrate storage.
We have established an effective protocol for long tem ERT by using the CNS minimal effective dosage 500U/kg. The use of this ERT regimen offered during a period of 3-6 months led to a more than 50% reduction of brain storage of alpha-Mannosidosis mice leading to a normalization of lysosomal function and a reduction of inflammatory parameters including microglia activation. We also successfully completed functional evaluations of different preclinical therapeutic trials. Bi-weekly injections of 500U/kg rhLAMAN during 14 weeks yielded partial improvements in exploratory activity and spatial learning & memory of alpha-mannosidosis mice (age of initiation ~ 3 months). Furthermore, we confirmed efficacy of enzyme replacement therapy to improve treadmill performance in these mice, which became evident during earlier short term experiments. Largely analogous results were obtained with the weekly treatment regimen of 500 U/kg rhLAMAN (age of initiation ~ 2 months). In the latter group we also observed a rescue in defective hippocampal long-term potentiation after long-term enzyme replacement therapy. Hereby we identified a possible mechanism for the beneficial effects on spatial learning in these mice.
Behavioral criteria for therapeutic evaluation were successfully confirmed for the novel immune-tolerant alpha-Mannosidosis mice during the first reporting period. During the second reporting period, we evaluated the behavioral efficacy of long-term ERT in different studies. Immune-tolerant alpha-Mannosidosis mice (age of initiation ~ 3 months) received bi-weekly intravenous injections of 500U/kg rhLAMAN for 14 weeks. During the last weeks of treatment functional efficacy was assessed by the following behavioral tests: Morris Water Maze (learning & memory), Open Field (emotional function & exploration) and Treadmill (motor performance). In another study, immune-tolerant alpha-Mannosidosis mice (age of initiation ~ 2 months) received weekly injections of 500U/kg rhLAMAN. These mice were treated for ~ 9 months in total. They received a first behavioral evaluation at the age of ~ 4.5 months. A second behavioral evaluation was performed at the age of ~ 8.5 months. Finally these mice also underwent electrophysiological evaluation (long-term potentiation) at the age ~ 10.5 months. For all behavioral experiments, these mice were compared to alpha-Mannosidosis and control mice receiving similar treatment regimens of intravenous saline.

Clinical Trials Phase 1-3 (WP3-6,9,10)

The preparation of the clinical trials were completed -During the last reporting period, the phase 3 preparation was completed and the protocol submitted and approved. Monitoring of phase 2b and 3 are completed (M15). During the first reporting period the WP4 work was concluded by qualitative and statistical review of data obtained and finally by the writing of a report of the phase 1 clinical trial. The clinical phase 2a “Dosing, evaluation of patients and reporting” was completed and phase 2b initiated after approval of Ethics committee and Competent authority (DKMA). The phase 2b was completed after all dosings of patients were completed and all evaluation and reporting conducted. Both the Phase 2a and 2b trials were extended, so the duration of this WP5 was much longer than originally planned and the resources used higher than expected. It was of outmost importance that the phase 3 dose and endpoints were defined on the best rational, why it was decided to prolong the phase 2a. After the Protocol Assistance meeting with the EMA, it was decided to change the placebo ratio and to expand the Phase 3 duration from 6mths to 12mths. The phase 3 application submission to the Ethics Committees were completed and the approval to initiate the trial was given in 6 countries. Dosing was conducted in 25 patients and all evaluations of patients was conducted in Copenhagen. The investigators report for Phase 3 has not been completed within the timeframe of the ALPHA-MAN project, since the last patient evaluation is scheduled to be performed 1 month after closure of the project, and unblinding of data and reporting awaits the trial completion. The Investigator report is though in preparation and data will be presented in articles after closure of this project work program.
The Pre-Trial regulatory filings for the IMPD were prepared and submitted as part of the authority evaluation af all clinical trials. The IMPD consists of a Clinical overview, a Drug Substance quality overview, appendix A.1 description of facilities, appendix A.2 adventitious agents safety evaluation, a Drug Product quality overview, a Pharmacology Written Summary, a Pharmacokinetics Written Summary and a Toxicology Written Summary. The Phase 3 protocol, including a synopsis, and Patient information/consent form were prepared and submitted to Ethic Committes and Competent Authorities in Belgium, Denmark, France, Germany, Spain and Sweden as the trial is a multinational EU trial. The monitoring of Phase 2b is completed and monitoring of Phase 3 is ongoing until the last-patient-last-visit is completed in May-2014. We consider it a major achievement of the ALPHA-MAN consortium that the phase 1 clinical trial could be conducted in the first reporting period without any safety concerns. This represented the first-in-man clinical trial with enzyme replacement therapy in alpha-Mannosidosis. This was a major achievement for patients with alpha-Mannosidosis as well as a prerequisite for conducting the following clinical phases with a focus on clinical effectiveness. There was a very high degree of compliance, with completion of almost all infusions in the Phase 2a and 2b trials, even though they were both prolonged. This is a major achievement in this logistically very demanding study. Also the efforts and investments of the families enrolled, that have resulted in such a high compliance cannot be underestimated. One very important achievement was the documentation, that Lamazym treatment was associated with a statistically significant improvement for a number of the biochemical and clinical efficacy endpoints, and that the minimum effective dose could be defined. From a safety point of view, Lamazym was well tolerated in the patients. The data suggests that ERT with Lamazym is an encouraging new treatment for patients with alpha-Mannosidosis. We also consider it a major achievement that the Phase 3 clinical trial could be conducted. It was very important that we were able to include 25 patients from european countries (Norway, Sweden, UK, Denmark, Netherland, Belgium, Poland, Germany, France and Spain). The effort and investments of the famillies enrolled in this challenging, time consuming study has been amazing, and the extremly high compliance of 98% conducted visits helped us ensure that the data quality is the best possible. It was decided that the evaluation of the chosen endpoints took place at partner 3 (CUH) in Copenhagen, Denmark in order to compare these evaluations directly with the evaluations performed in connection with WP4 and WP5. After discussion with the European Medicines Agency (EMA) it has been decided to change the placebo:active ratio from 33% to 40%.
The investigators report for Phase 3 has not been completed within the timeframe of the ALPHA-MAN project, as the last patient evaluation is scheduled to be performed 1 month after closure of the project, and unblinding of data and reporting awaits the trial completion. The reason for this deviation from the project work program is the prolonged Phase 2 trial, the extension of the phase 3 trial, the increased number of patients participating in the phase 3 trial and because the recruitment of patients to the Phase 3 took longer than expected. The Investigator report is in preparation and data will be presented in articles after closure of this project work program.
All clinical data that have been collected during the clinical trials. This involves four main areas of expertise and work: statistics, data management, reporting and pharmacovigilance. Work has progressed successfully in all four areas along with the progress and completion of the clinical phase trials. Industry level databases have been defined for the trials, data have been retrieved and validated, data have been analysed statistically and integrated clinical reports have been finalised.
Study set-up, including tailor-made study materials, sampling kits and the LIMS data base set-up was performed prior to the study initiation. Biochemistry, haematology and coagulation analyses were performed on all safety samples. Assay of 172 rhLAMAN PK samples from the rhLAMAN-05 study.

Analysis of alpha-Mannosidosis mutations (WP7)
Novel mutant MAN2B1 expression constructs have been created by in vitro mutagenesis and subsequently assayed for enzyme activity, and intracellular processing and localization. All missense mutations have been reanalyzed for intracellular localization as previous methods have been replaced by confocal microscopy. In this period all missense mutations have been subjected to 3 dimensional remodeling and presented with an improved and standardized lay-out. The alpha-mannosidosis mutation database (amamutdb.no) has been further developed by implementing new layout and new applications which allow the user to assess database statistics and to retrieve data and information across datasets. Molecular and clinical data have been subjected to thorough quality assurance. A further improvement of the curator function has been carried out. To a large extent this facilitates data entry and quality assurance. The latest version of amamutdb.no can be accessed at: to be opened in Chrome or Mozilla Firefox. The results obtained provide a comprehensive picture of the genetic etiology of alpha-mannosidosis in Europe. The mutation database now includes genetic, biochemical and/or clinical data on more than 240 patients from 42 countries. The database contains 166 sequence variants of which 150 are thought to be disease causing (including 4 mutations which occur in animal models) and 16 are thought to be benign. Missense mutations have been reinvestigated with regard to intracellular localization and their effect on the MAN2B1 3-dimensional structure. New and updated figures on intracellular localization and 3-D models have been included.
Analysis of Oligosaccharides (WP8)
We have succesfully applied the analytical method detection for the detection, characterization and quantification of Mannose-2 oligosaccharide (Man2) present in serum and cerospinal fluids /CSF) collected from patients with alpha-Mannosidosis before and after treatment with rhLAMAN in all the clinical trials. Analysis of oligosaccharides in urine, serum, tissues and cerebrospinal fluids from primarily patients, as well as in various organs from mice, has been performed to evaluate the efficiacy of chronic ERT treatment. The results demonstrated that the ERT lowered the 2-mannose in the body fluids from patients treated with ERT.










Potential Impact:
The ultimate impact of the project was to establish a therapeutic strategy to treat the rare disorder alpha-Mannosidosis. Currently no specific treatment for alpha-Mannosidosis exists. Present treatments are confined to be supportive and symptomatic. Prevention of the disease is restricted to prenatal diagnosis and carrier identification. The results obtained within the different objectives described above are of great value for the introduction of recombinant human lysosomal alpha-mannosidase for ERT in patients. On the one hand, we were able to evaluate the benefit of alpha mannosidase ERT in the mouse model and we obtained a suitable protocol for long term treatment of this lysosomal storage disorder. Importantly, the correction of storage could be followed over time in visceral organs but also in the central nervous system. An improvement of the brain associated symptoms was followed by detailed behavioral monitoring of treated and non treated mice, respectively. On the other hand, a systematic clinical and diagnostic natural history study of the disease in human patients enabled us to define the critical parameters and hallmarks of the disease which could be used in phase I and phase II and phase III clinical trials to evaluate the efficacy of the ERT in patients.

ALPHA-MAN has been of substantial importance to the application for marketing and approval of Lamazym as a therapeutic agent for ERT in alpha-Mannosidosis patients. All the goals of ALPHA-MAN added new important informations which paved the way to realise a specific and evidence based cure of this still untreatable disease.
The major technological prospect of this research is the achievement and introduction of a medical therapy for treating alpha-Mannosidosis. This research is intended to decrease the economical burden, and to improve the prospects of a new medicament, to the point where there is an incentive among biotechnological companies to produce this drug.


Zymenex (Participant 2) has an interest in obtaining proprietary patents on inventive steps of manufacturing procedures, new methods and the drug (recombinant human alpha-Mannosidase), to aid the company in developing a therapy for alpha-Mannosidosis and thus preventing third parties from commercially exploiting the product without authorisation. This protection improves the company's possibilities for presenting a business case to investors and helps secure that the product will have the potential to reach the market .

List of Websites:
https://web.archive.org/web/20170808214806/http://www.alpha-man.eu/index.htm

Contact details for website:
Prof. Dr. Paul Saftig
Dr. Michael Pachta
Biochemisches Institut
Christian-Albrechts-Universität Kiel
Olshausenstr. 40
D-24098 Kiel
Germany