{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:59270"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:59270","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Generation and characterisation of plant produced recombinant antibodies specific to LHRH for treatment of sex hormone dependent diseases","abstract":"Leutinising hormone releasing hormone (LHRH) is a hypothalamic neurohormone that plays a pivotal role in the endocrine control of reproduction. Acting through its high affinity receptors on pituitary gonadotrophs, it regulates the secretion of gonadotropins. Manipulation of the neuroendocrine cascade by LHRH intervention leads to the down regulation of sex steroid levels resulting in a state of pharmacological castration, a physiological state useful for treatment of sex hormone dependent diseases. The neuroendocrine axis can be obliterated by synthetic LHRH analogues or by neutralising anti-LHRH antibodies. While the LHRH analogues require frequent dose administration and are expensive, active vaccination against LHRH is limited by the variation in the immune response from patient to patient and the lag period in the build-up of effective antibody titres. The aim of this project was the generation of recombinant anti-LHRH antibodies and their expression in plants for use in passive immunisation for treatment of sex hormone dependent disorders. This thesis reports the construction of a single chain variable fragment, a diabody and a mouse/human full-length chimeric antibody by recombinant DNA technology. The variable domains that constituted these antibody molecules were derived from a high affinity anti-LHRH mouse mAb. The antibodies were transiently expressed in tobacco leaves by vacuum-assisted agro-infiltration and purified by affinity chromatography. While the chimeric antibody was expressed at moderately high levels in tobacco leaves the expression levels of the scFv and diabody fragments was found to be low. All three affinity purified antibody molecules were functional, pure and intact as determined by ELISA and SDS-PAGE analysis. The equilibrium constants of the plant expressed chimeric rAb and the parental murine mAb, as measured by SPR using BIAcore, were found to be indistinguishable. Incubation of chimeric rAb and the mouse mAb in human serum in vitro revealed high stability of both the mouse mAb and the chimeric antibody at the physiological temperature. Antibody activity after 32 days at 37°C in human serum was determined to be 70% for chimeric rAb and 60% for mouse mAb. Both the anti-LHRH mAb and the plant derived chimeric antibody were shown to be bio-effective. The antibodies caused an arrest in the estrus cycle of regularly cycling rodents. The duration of the estrus cycle disruption was found to be related to the dose of the antibody administered. A set of related peptide motifs were also isolated from a phage displayed random 12-mer-peptide library that unambiguously bound to the LHRH specific chimeric mAb. The isolated peptides are mimotopes, as they do not show any similarity with LHRH at the primary structural level. Furthermore, binding analysis of a few LHRH variants with the murine mAb revealed that the antibody also recognizes two other isoforms of LHRH reported to be found in humans.","abstract_html":"Leutinising hormone releasing hormone (LHRH) is a hypothalamic neurohormone that plays a pivotal role in the endocrine control of reproduction. Acting through its high affinity receptors on pituitary gonadotrophs, it regulates the secretion of gonadotropins. Manipulation of the neuroendocrine cascade by LHRH intervention leads to the down regulation of sex steroid levels resulting in a state of pharmacological castration, a physiological state useful for treatment of sex hormone dependent diseases. The neuroendocrine axis can be obliterated by synthetic LHRH analogues or by neutralising anti-LHRH antibodies. While the LHRH analogues require frequent dose administration and are expensive, active vaccination against LHRH is limited by the variation in the immune response from patient to patient and the lag period in the build-up of effective antibody titres. The aim of this project was the generation of recombinant anti-LHRH antibodies and their expression in plants for use in passive immunisation for treatment of sex hormone dependent disorders. This thesis reports the construction of a single chain variable fragment, a diabody and a mouse/human full-length chimeric antibody by recombinant DNA technology. The variable domains that constituted these antibody molecules were derived from a high affinity anti-LHRH mouse mAb. The antibodies were transiently expressed in tobacco leaves by vacuum-assisted agro-infiltration and purified by affinity chromatography. While the chimeric antibody was expressed at moderately high levels in tobacco leaves the expression levels of the scFv and diabody fragments was found to be low. All three affinity purified antibody molecules were functional, pure and intact as determined by ELISA and SDS-PAGE analysis. The equilibrium constants of the plant expressed chimeric rAb and the parental murine mAb, as measured by SPR using BIAcore, were found to be indistinguishable. Incubation of chimeric rAb and the mouse mAb in human serum in vitro revealed high stability of both the mouse mAb and the chimeric antibody at the physiological temperature. Antibody activity after 32 days at 37°C in human serum was determined to be 70% for chimeric rAb and 60% for mouse mAb. Both the anti-LHRH mAb and the plant derived chimeric antibody were shown to be bio-effective. The antibodies caused an arrest in the estrus cycle of regularly cycling rodents. The duration of the estrus cycle disruption was found to be related to the dose of the antibody administered. A set of related peptide motifs were also isolated from a phage displayed random 12-mer-peptide library that unambiguously bound to the LHRH specific chimeric mAb. The isolated peptides are mimotopes, as they do not show any similarity with LHRH at the primary structural level. Furthermore, binding analysis of a few LHRH variants with the murine mAb revealed that the antibody also recognizes two other isoforms of LHRH reported to be found in humans.","abstract_has_math":false,"creators":["Nath, Richa"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Fischer, Rainer"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2003,"date_issued":"2003","date_published":"2003","updated_at":"2026-07-30T19:42:39Z","subjects":["info:eu-repo/classification/ddc/580","Pflanzen (Botanik)","Recombinant antibodies","transgenic plants","molecular farming","molecular biotechnology","scFv","chimeric antibody"],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121071%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121071%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121071%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/59270","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Fischer, Rainer"]},{"key":"dc:creator","label":"Author","values":["Nath, Richa"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2003"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/doi/10.18154/RWTH-CONV-121071","info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-7433"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/580","Pflanzen (Botanik)","Recombinant antibodies","transgenic plants","molecular farming","molecular biotechnology","scFv","chimeric antibody"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/59270","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121071%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Leutinising hormone releasing hormone (LHRH) is a hypothalamic neurohormone that plays a pivotal role in the endocrine control of reproduction. Acting through its high affinity receptors on pituitary gonadotrophs, it regulates the secretion of gonadotropins. Manipulation of the neuroendocrine cascade by LHRH intervention leads to the down regulation of sex steroid levels resulting in a state of pharmacological castration, a physiological state useful for treatment of sex hormone dependent diseases. The neuroendocrine axis can be obliterated by synthetic LHRH analogues or by neutralising anti-LHRH antibodies. While the LHRH analogues require frequent dose administration and are expensive, active vaccination against LHRH is limited by the variation in the immune response from patient to patient and the lag period in the build-up of effective antibody titres. The aim of this project was the generation of recombinant anti-LHRH antibodies and their expression in plants for use in passive immunisation for treatment of sex hormone dependent disorders. This thesis reports the construction of a single chain variable fragment, a diabody and a mouse/human full-length chimeric antibody by recombinant DNA technology. The variable domains that constituted these antibody molecules were derived from a high affinity anti-LHRH mouse mAb. The antibodies were transiently expressed in tobacco leaves by vacuum-assisted agro-infiltration and purified by affinity chromatography. While the chimeric antibody was expressed at moderately high levels in tobacco leaves the expression levels of the scFv and diabody fragments was found to be low. All three affinity purified antibody molecules were functional, pure and intact as determined by ELISA and SDS-PAGE analysis. The equilibrium constants of the plant expressed chimeric rAb and the parental murine mAb, as measured by SPR using BIAcore, were found to be indistinguishable. Incubation of chimeric rAb and the mouse mAb in human serum in vitro revealed high stability of both the mouse mAb and the chimeric antibody at the physiological temperature. Antibody activity after 32 days at 37°C in human serum was determined to be 70% for chimeric rAb and 60% for mouse mAb. Both the anti-LHRH mAb and the plant derived chimeric antibody were shown to be bio-effective. The antibodies caused an arrest in the estrus cycle of regularly cycling rodents. The duration of the estrus cycle disruption was found to be related to the dose of the antibody administered. A set of related peptide motifs were also isolated from a phage displayed random 12-mer-peptide library that unambiguously bound to the LHRH specific chimeric mAb. The isolated peptides are mimotopes, as they do not show any similarity with LHRH at the primary structural level. Furthermore, binding analysis of a few LHRH variants with the murine mAb revealed that the antibody also recognizes two other isoforms of LHRH reported to be found in humans."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University 159 S. : Ill., graph. Darst. (2003). doi:10.18154/RWTH-CONV-121071 = Aachen, Techn. Hochsch., Diss., 2003"]},{"key":"dc:title","label":"Title","values":["Generation and characterisation of plant produced recombinant antibodies specific to LHRH for treatment of sex hormone dependent diseases"]}]}],"canonical_facts":{"dc:contributor":["Fischer, Rainer"],"dc:coverage":["DE"],"dc:creator":["Nath, Richa"],"dc:date":["2003"],"dc:description":["Leutinising hormone releasing hormone (LHRH) is a hypothalamic neurohormone that plays a pivotal role in the endocrine control of reproduction. Acting through its high affinity receptors on pituitary gonadotrophs, it regulates the secretion of gonadotropins. Manipulation of the neuroendocrine cascade by LHRH intervention leads to the down regulation of sex steroid levels resulting in a state of pharmacological castration, a physiological state useful for treatment of sex hormone dependent diseases. The neuroendocrine axis can be obliterated by synthetic LHRH analogues or by neutralising anti-LHRH antibodies. While the LHRH analogues require frequent dose administration and are expensive, active vaccination against LHRH is limited by the variation in the immune response from patient to patient and the lag period in the build-up of effective antibody titres. The aim of this project was the generation of recombinant anti-LHRH antibodies and their expression in plants for use in passive immunisation for treatment of sex hormone dependent disorders. This thesis reports the construction of a single chain variable fragment, a diabody and a mouse/human full-length chimeric antibody by recombinant DNA technology. The variable domains that constituted these antibody molecules were derived from a high affinity anti-LHRH mouse mAb. The antibodies were transiently expressed in tobacco leaves by vacuum-assisted agro-infiltration and purified by affinity chromatography. While the chimeric antibody was expressed at moderately high levels in tobacco leaves the expression levels of the scFv and diabody fragments was found to be low. All three affinity purified antibody molecules were functional, pure and intact as determined by ELISA and SDS-PAGE analysis. The equilibrium constants of the plant expressed chimeric rAb and the parental murine mAb, as measured by SPR using BIAcore, were found to be indistinguishable. Incubation of chimeric rAb and the mouse mAb in human serum in vitro revealed high stability of both the mouse mAb and the chimeric antibody at the physiological temperature. Antibody activity after 32 days at 37°C in human serum was determined to be 70% for chimeric rAb and 60% for mouse mAb. Both the anti-LHRH mAb and the plant derived chimeric antibody were shown to be bio-effective. The antibodies caused an arrest in the estrus cycle of regularly cycling rodents. The duration of the estrus cycle disruption was found to be related to the dose of the antibody administered. A set of related peptide motifs were also isolated from a phage displayed random 12-mer-peptide library that unambiguously bound to the LHRH specific chimeric mAb. The isolated peptides are mimotopes, as they do not show any similarity with LHRH at the primary structural level. Furthermore, binding analysis of a few LHRH variants with the murine mAb revealed that the antibody also recognizes two other isoforms of LHRH reported to be found in humans."],"dc:identifier":["https://publications.rwth-aachen.de/record/59270","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121071%22"],"dc:language":["eng"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/doi/10.18154/RWTH-CONV-121071","info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-7433"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University 159 S. : Ill., graph. Darst. (2003). doi:10.18154/RWTH-CONV-121071 = Aachen, Techn. Hochsch., Diss., 2003"],"dc:subject":["info:eu-repo/classification/ddc/580","Pflanzen (Botanik)","Recombinant antibodies","transgenic plants","molecular farming","molecular biotechnology","scFv","chimeric antibody"],"dc:title":["Generation and characterisation of plant produced recombinant antibodies specific to LHRH for treatment of sex hormone dependent diseases"],"dc:type":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]},"updated_at":"2026-07-30T19:42:39Z"}