Messung von L-Asparaginase-Antikörper-Titern von pädiatrischen Patienten mit akuter lymphatischer Leukämie der Nordic Society of Pediatric Hematology and Oncology unter Einsatz eines epitopspezifischen ELISA

L-Asparaginase wird seit mehr als etwa 30 Jahren in der Behandlung der akuten lymphatischen Leukämie sowie einiger Non-Hodgkin-Lymphome im Kindes- und Erwachsenenalter eingesetzt. Die zytotoxische Wirksamkeit des Enzyms beruht auf der Depletion der L-Asparagin-Serumkonzentration durch Hydrolyse...

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Bibliographic Details
Main Author: Angermeier, Stefan
Contributors: Röhm, Klaus-Heinrich (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2008
Online Access:PDF Full Text
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L-asparaginase has been an essential part in the treatment schedules of acute lymphoblastic leukemia and some non-hodgkin-lymphoms in children and adults for more than 30 years. The cytotoxic mechanism of the enzyme is based on the depletion of L-asparagine by hydrolysis of the amino acid L-asparagine into L-aspartic acid and ammonia. The tumor cells lack sufficient amounts for themselves for synthesizing, resulting in an impairment of the protein biosynthesis and finally in cell death. Because of the bacterial origin immunological reactions are common side effects under a therapy with L-asparaginase. The formation of specific antibodies by sensitization with the protein may become clinically manifest in different ways. Nearly 30% of the patients suffer from hypersensitivity reactions, which presents themselves in a wide spectrum from local irritations to systemic anaphylaxis. In addition some patients will develop a faster decrease of the enzymatic activity. Due to the absence of clinical symptoms this pheno¬menon is called “silent inactivation”. This process influences the pharmacokinetic and finally the therapeutic outcome significantly. Currently a determination method of antibodies against L-asparaginase is not available in clinical practice. The use of the complete L-asparaginase protein results in cross reactions, which limit the information about the appearance of a relevant immunological reaction. Because of its low specificity and sensitivity of the assays drug-monitoring programs measure the enzymatic activity for therapy control. For improvement of the prediction of hypersensitivity reactions and “silent inactivation” a project was started with the aim of developing an epitope-specific assay for the detection of L-asparaginase antibodies. In the present study six synthetic peptides of L-asparaginase were used as antigens in ELISA instead of the complete protein. A screening for anti¬bodies binding was performed by using sera of 42 patients with and without “silent inactivation”, which were kindly made available by the NOPHO-ALL 2000 group, a study group for the treatment of ALL in children in Scandinavian countries. A correlation between the binding of specific antibodies against two of the six peptides and a “silent inactivation” could be observed. In the studied collective patients with a fast decrease of the enzyme activity (n=19) had a significantly more frequent binding of antibodies to the fragment with amino acids 1-136 than the control group (n=14) at the end of reinduction. A frequent, but not significantly binding to the fragment with amino acids 214-326 could be demonstrated. A significant higher increase of antibody titers from induction to reinduction was shown for the “silent inactivation” group versus the “therapeutic activity” for the fragment with amino acids 1-136 and the complete L-asparaginase protein. The amino acids 1-136 and 214-326 correspond to the N- and C-terminal part of the L-asparaginase molecule and are situated near the active center of the enzyme. A binding of antibodies may affect the enzymatic activity and may provide us with an explanation for the action of “silent inactivation”. Patients with an enzyme activity in a therapeutic range (n=23) showed only a marginal binding of antibodies. Cross reactions also impaired the method of the epitop-specific assay. The influence of cross reactions by antibodies measurement to the complete L-asparaginase protein was less than in other published works. By conducting more prospective studies an optimization of an epitope-specific assay and the detection of clinically relevant epitopes could be achieved. In the setting of a rational drug monitoring of L-asparaginase therapy in acute lymphoblastic leukemia the measurement of the enzyme activity is at first irreplaceable.